WO2013028051A1 - Composition comprising sandoricum koetjape extracts and uses thereof - Google Patents

Composition comprising sandoricum koetjape extracts and uses thereof Download PDF

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WO2013028051A1
WO2013028051A1 PCT/MY2012/000009 MY2012000009W WO2013028051A1 WO 2013028051 A1 WO2013028051 A1 WO 2013028051A1 MY 2012000009 W MY2012000009 W MY 2012000009W WO 2013028051 A1 WO2013028051 A1 WO 2013028051A1
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acid
koetjapic
composition
cells
cancer
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PCT/MY2012/000009
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French (fr)
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Amin Malik Shah Abdul Majid
Khalid M. ABU-SALAH
Zhari ISMAIL
Mohamed Khadeer AHAMED
Zeyad Daoud NASSAR
Abdalrahim F.A AISHA
Mahibub MAHAMADSA
Aman Shah ABDUL MAJID
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Universiti Sains Malaysia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/58—Meliaceae (Chinaberry or Mahogany family), e.g. Azadirachta (neem)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • VEGF vascular endothelial growth factor
  • the hypoxic state in the tumor spring forth oxygen free radicals which in turn activates vascular endothelial growth factor (VEGF) triggering the angiogenesis event.
  • VEGF had been regarded as a heparin binding angiogenic growth factor exhibiting high specificity for endothelial cells.
  • VEGF is responsible triggering the various steps in the angiogenesis cascade such as proliferation, migration and cell survival.
  • the tumor regression and inhibition can be achieved by deactivating VEGF activity via neutralizing antibodies or by the introduction of dominant negative VEGF receptors into endothelial cells of tumor-associated blood vessels. Mopping up free radicals with high level of anti-oxidants can also affect transcription of the VEGF gene. This highlights the central role of VEGF in tumor angiogenesis.
  • Triterpenes are a class of phytochemicals that have wide pharmacological activity.
  • Koetjapic acid (KA) is a seco-A-ring oleanene triterpene, isolated and reported by Kaneda et al., (1992) for the first time from Sandoricum koetjape.
  • Sandoricum koetjape is a traditional plant belonging to the family of Meliaceae. It is native to Southeast Asian countries, including Malaysia and Philippines. In Malaysia, it is locally known as santol. The tree is a medium-sized with edible fruit.
  • Colorectal cancer less formally known as bowel cancer, is a cancer characterized by neoplasia in the colon, rectum, or vermiform appendix. Colorectal cancers start in the lining of the bowel. If left untreated, it can grow into the muscle layers underneath, and then through the bowel wall. Most begin as a small growth on the bowel wall: a colorectal polyp or adenoma. These mushroom-shaped growths are usually benign, but some develop into cancer over time. Localized bowel cancer is usually diagnosed through colonoscopy. Colorectal cancer is the third most commonly diagnosed cancer in the world, but it is more common in developed countries.
  • koetjapic acid exhibits anti-angiogenic property by inhibiting the in vivo neovascularization in chick embryo chorioallantoic membrane (CAM) and ex vivo microvessel sprouting in rat aortic explants.
  • Koetjapic acid inhibits angiogenesis by down regulation of VEGF signaling pathway by inhibiting the VEGF induced HUVECs proliferation, migration and differentiation, and by inhibiting the synthesis of the principal angiogenic mitogen, VEGF.
  • the present invention provides NF- ⁇ up-regulation and MAPK/JNK, MAPK/ERK, HIF, MYC/MAX and Wnt down-regulation composition comprising koetjapic acid as active ingredient without other active ingredients.
  • the composition of the present invention can be used for the treatment or prevention of diseases derived from under-expression of NF- ⁇ gene and or over- expression of MAPK/JNK, MAPK/ERK, HIF, MYC/MAX and Wnt genes or pathways.
  • the present invention provides an anti-angiogenic and anti-tumorigenic composition comprising koetjapic acid for pharmaceutical, dietetic, health supplement and or nutraceutical uses.
  • the present invention is also directed to a composition
  • a composition comprising koetjapic acid for anti-angiogencially and or apoptotically suppress or reduce the tumorigenic cancer.
  • the composition additionally comprises for the treatment of the abnormality caused by at least one gene oncogene or the genes directly or indirectly associated with angiogenesis or cancer.
  • the composition additionally comprises at least one angiogenesis or cancer related genes such as NF- B, MAPK/JNK, MAPK/ERK, HIF, MYC/ AX and Wnt.
  • the composition may be a pharmaceutical composition for anti-angiogenic or antitumor activities along with any classical chemotherapeutics against cancer.
  • a and “an” are used to refer to both single and a plurality of objects.
  • "koetjapic acid” refers to a seco-A-ring oleanene triterpene, which can be systematically characterized as "(3R,4a 4bS,7S,8S,10bS,12aS)-7-(2- carboxyethyl)-3,4b,7,10b,12a-pentamethyl-8-(prop-1-en-2-yl)- 1 ,2,3,4,4a,4b,5,6,7,8,9,10,10b,1 1 ,12,12a-hexadecahydrochrysene-3-carboxylic acid” or it can also be systematically named as "3-((1 S,2S,4BS,6Ar,10bS)- 1 ,4b,6a,9,9,10b,-hexamethyl-2-(prop-1-en-2-yl)-
  • compositions in their alternate forms may be used alone or in combination to provide an anti-angiogenic or anti- tumorigenic herbal product or synthetic or semi-synthetic medicine, which when given to a patient, results in the preventive or therapeutic effect against anti-tumor or angiogenesis-related aliments.
  • administration can be made via any accepted systemic delivery system, for example, via oral route or parenteral route such as intravenous, intramuscular, intradermal, subcutaneous or percutaneous route, or vaginal, ocular or nasal route, in solid, semi-solid or liquid dosage forms, such as for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, cream, gel, implant, patch, pessary, aerosols, collyrium, emulsions or the like, preferably in unit dosage forms suitable for easy administration of fixed dosages.
  • the pharmaceutical compositions will include a conventional carrier or vehicle and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, and so on.
  • the tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of winter
  • subject is a vertebrate, preferably a mammal, more preferably a human.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • Figure 4 shows koetjapic acid inhibition of proliferation of four isogenic human tumor cell lines (HCT 116, MCF7, MDA-MB-231 , and Hep G2) and normal HUVECs in dose dependent manner.
  • HCT 116, MCF7, MDA-MB-231 , and Hep G2 isogenic human tumor cell lines
  • HUVECs normal HUVECs in dose dependent manner.
  • Figure 8 shows koetjapic acid inducing DNA fragmentation.
  • HCT 1 1 16 cells were treated with 0.1 % ethanol (negative control) betulinic acid at 20 ⁇ / ⁇ (positive control) and the indicated concentrations of koetjapic acid for 24 hours. Both floating and adherent cells were collected, DNA was extracted and electrophoresed on a 1 .2 % agarose gel were stained with ethidium bromide and photographed;
  • Figure 10 shows signalling pathways influenced by koetjapic acid. Significant decrease in cancer reporter gene activities was noted for pro-proliferative pathways including Wnt, c-Myc, hypoxia, MAPK/ERC and MAPK /JNK, and significant up-regulation of NF- ⁇ pathway can be seen in koetjapic acid (25 pg/mi). No significant changes detected in Notch, P53, TGF& and pRb-E2F. Error bars indicate standard deviations from mean. * P > 0.05, ** P > 0.01 and *** P > 0.001 ;
  • composition comprising koetjapic acid of the current invention is used as an anti-angiogenic and anti-tumorigenic agent for the treatment or prevention of angiogenesis-dependent pathologies and colorectal cancer respectively, for pharmaceutical, preventive or chemotherapeutical, nutraceutical, or health supplement, dietetic use.
  • n-Hexane or the various proportional ratios of n-Hexane with chloroform, or acetone, or any other organic solvent is added to 1 kg of dried S. koetjape stem bark powder.
  • the mixture is allowed to extract at a temperature ranging from 40 to 60°C, for a period ranging from 10 minutes to 48 hours or more.
  • cold extraction procedure can also be followed at room temperature.
  • the extraction process may be repeated 2 to 3 times with other solvents (chloroform, ethyl acetate, dichloro ethane, ketone, etc.).
  • the resulting extract is concentrated to obtain S. koetjape extract.
  • the asymmetric unit of title koetjapic acid consists of one koetjapic acid molecule and half a molecule of disordered chloroform solvent.
  • the koetjapic acid contains fused four-ring system, A/B/C/D (see Fig. 1 ).
  • the A/B, B/C and C/D junctions adopt E/trans/cis configuration, respectively.
  • Table 1 depicts the basic Crystal Data obtained from the crystallographic analysis.
  • Refinement Refinement of F 2 against ALL reflections.
  • the weighted R-factor wR and goodness of fit S are based on F 2
  • conventional f?-factors R are based on F, with F set to zero for negative F 2 .
  • the threshold expression of F 2 > o(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement.
  • R-factors based on F 2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger.
  • the present inventors also studied the apoptotic morphological events occurring in the nuclei after incubation of HCT 1 16 cells with koetjapic acid. No significant morphological changes were observed in nuclei incubated in the absence of koetjapic acid. At higher concentrations of koetjapic acid 30 g/ml the nuclei displayed the typical apoptotic changes in the chromatin structure appearing remarkably condensed at the nuclear periphery. Chromatin condensed progressively, in a dose dependent manner (see Fig.
  • the mitochondrial membrane potential in HCT 1 16 cells was evaluated by visualizing the uptake of the lipophilic cation dye rhodamine into mitochondria.
  • the loss of mitochondrial membrane potential ( ⁇ ) is a hallmark for apoptosis [32].
  • the rhodamine 123 uptake by the cells increases and the florescent signal intensifies exponentially.
  • Our results showed an obvious intensification of fluorescence in cells treated with koetjapic acid (20 g/ml) compared with control samples, which suggests a loss in mitochondrial membrane potential.
  • the fluorescent intensity increased with treatment duration (see Fig. 9) the effect of koetjapic acid on mitochondrial membrane potential.
  • Koetjapic acid modulated the gene expression in HCT 1 16
  • koetjapic acid To characterize the anti-angiogenesis activity of koetjapic acid, we first determined whether koetjapic acid inhibited the growth factors induced tube formation of endothelial cells on Matrigel (see Fig. 13G) depicted a dose-dependent inhibition of tube formation by HUVECs. Endothelial cells formed tube-like networks (see Fig. 13A) within 6 hr, which might, in part, reflect the process of angiogenesis. At a concentration of 40 pg/ml (see Fig. 13E), the koetjapic acid absolutely abrogated endothelial tube formation by reducing the tube-like structure both in width and in length.
  • PBS Phosphate buffered saline
  • PS penicillin/streptomycin
  • Human umbilical vein endothelial cell line was purchased from ScienCeli, USA. HUVECs were maintained in ECM medium supplemented with 5% HIFBS, 1 % PS and 1 % ECGS. Human colorectal carcinoma (HCT 1 16), high invasive human adenocarcinama cells (MCF 7), less invasive breast ductal carcinoma cells (MDA-MB-231 ) and Hep G2 (liver cancer cell line) were purchased from American type culture collection (Rockvill, MD, USA). HCT 1 16 and cell lines were maintained in RPMI 1640 containing 10% HIFBS and 1 % PS. MCF7, MDA- MB-231 and Hep G2 were cultured in DMEM supplemented with 10% HIFBS and 1 % PS. Cells were cultured in a 5% C0 2 -humidifed atmosphere at 37 °C.
  • the koetjapic acid was crystallized using chloroform to yield colourless prism-shaped crystals in the amount of 400 mg.
  • the melting point of the compound was recorded in a Toshniwal melting point apparatus.
  • the UV spectrum was recorded on a Perkin Elmer Lambda 25 UV spectrophotometer.
  • the IR Spectrum was recorded with KBr pellets on a Thermo nexus FT-IR spectrophotometer.
  • the 1 H NMR spectrum of the compound was taken on a Bruker AMX (300 MHz) Spectrometer using CDCI 3 as solvents.
  • X-ray analysis was studies using Bruker APEX II DUO CCD area- detector diffractometer.
  • a clonogenic assay was carried out according to (Franken et al., 2006). In brief, 1000 cells/well within single cell suspension were plated in six well plates. After cells attachment, cells were treated with koetjapic acid (12.5, 25, 50 and 75 Mg/ml), betulinc acid 20 9/ ⁇ as a positive control or 1 % ethanol as a negative control. After 48 h of treatment, media was removed, cells were washed twice with PBS and new fresh medium was added. After 7 days the cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. The colonies of >50 cells were counted. Plating efficiency (PE) which is the ratio of the number of colonies which grown from untreated cells to the number of cells seeded was calculated using this formula:
  • HCT 1 16 cells were subjected to Caspase 3/7, 8, 9 activities measurement with Caspase-Glo assay kit (Promega, USA).
  • Caspase-Glo assay kit Promega, USA.
  • 100 ⁇ of Caspase-Glo reagent was added to each well. The plate was then incubated for 30 minutes at room temperature. The luminescence which is proportional to net caspases activities was measured in a plate-reading luminometer (HIDEX, Finland). The experiments were performed in triplicate.
  • HCT 1 16 cells (5 x 10 ) were treated with various concentrations of koetjapic acid (KA) for 24 hours.
  • DNA was extracted by suspending cells in a lysis buffer provided with Wizard ® SV Genomic DNA Purification kit (Promega ,USA ). DNA pellets were electrophoresed for 2 hours at 100 V in 1.2% agarose gel. The gel was stained with ethidium bromide, and the DNA fragments were visualized under ultraviolet light. Determination of nuclear condensation by Hoechst 33342 stain
  • luciferase activity was measured using the Dual Luciferase Assay system (Promega , USA).
  • the Firefly and Renilla activities represented the experimental and normalizing reporters respectively. Reporter activity was performed in triplicate and the result was presented as a mean of a fold change ⁇ SD for each pathway.
  • Symmetry codes (i) -x+1 , -y+2, z.
  • KA Anti-proliferative efficacy of koetjapic acid
  • Cytotoxic potency of koetjapic acid (KA) was evaluated using MTT assay on HUVEC, HCT 1 16, MCF7, MDA-MB-231 and Hep G2 cell lines.
  • the inhibitory effect of KA on HCT 1 16 was comparable with the positive controls used. Table 1 depicts the IC 50 values of all tested compounds on the indicated cell lines. iii. Colony formation assay
  • KA KA induced inhibition of cell proliferation was due to induction of apoptosis.
  • KA induced the appearance of caspases activities in HCT 1 16 cells in presence of Caspase-Glo reagent. From the results it is conspicuous that, KA activates the initiator caspases, i.e., caspases 8 and 9. The activity of the initiator caspases in treated cells was about 3 fold higher than the control. Furthermore, KA was found to be more specific for the induction of caspase 8 activity, since the induction of caspases 9 was less significant than the caspase 8.
  • Fig. 6 illustrates the induction of caspases activity by koetjapic acid. v. DNA fragmentation Induced by koetjapic acid
  • Figs. 14A and 14B Images of two chorioallantoic membranes are shown in Figs. 14A and 14B.
  • the vasculature pattern formed by the blood vessels in CAM of control group treated with vehicle was normal.
  • the primary, secondary and tertiary vessel with the dendritic branching pattern, which is characteristic of CAMs, was well established in control CAMs and can be seen clearly in Fig. 14A.
  • the 100 pg KA treated CAM (1 mg/disc) showed the distorted architecture in vasculature, as shown in Fig. 14B.
  • KA inhibited microvessel formation in rat aorta, and thwart endothelial cell proliferation and migration which all essential for angiogenesis. It is noteworthy to mention that Wnt signaling is also involved in the regulation of endothelial cell proliferation and migration during angiogenesis, and HIF overexpression is associated with pathologic angiogenesis. It is likely that the observed ex vivo and in vitro anti-angiogenic potencies observed in the present invention are the consequence of koetjapic acid (KA) induced Wnt and HIF down-regulation. The antiangiogenic activity of koetjapic acid (KA) is further supported by the in vivo studies employing fertilized chicken embryo (CAM) where compound caused marked inhibition in blood vessel formation.
  • CAM fertilized chicken embryo
  • Aspirin a non-steroidal anti-inflammatory drug, induces apoptosis in colon cancer cells by upregulating the NF- ⁇ signalling pathway, koetjapic acid (KA) is reported to have an anti-inflammatory property, and most probably, similar to the aspirin it may stimulate NF- ⁇ through nuclear translocation of NF- ⁇ complexes.
  • KA koetjapic acid

Abstract

The present invention discloses a composition consisting essentially of an amount of a compound effective for inhibiting colorectal cancer cell growth, the compound having the following structure isolated and purified from Sandoricum koetjape plant extract.

Description

COMPOSITION COMPRISING SANDORICUM KOETJAPE EXTRACTS AND
USES THEREOF
FIELD OF THE INVENTION
The present invention relates to extracts from a plant called Sandoricum koetjape, their usages and functions in the treatment of cancer, and method for their preparation. BACKGROUND OF THE INVENTION
Angiogenesis is a process of new blood vessel development orchestrated by a range of angiogenic factors and inhibitors. This process is tightly regulated and self limiting in some cases such as wound healing, normal growth process and reproductive function. In contrast, when this process is deregulated, diseases such as cancer, rheumatoid arthritis, obesity, diabetic blindness can form. Angiogenesis plays an important role in cancer growth without which, tumors will be unable to expand beyond 1 to 2 mm3. Cancer cells within the tumor will then use the newly formed blood vessels as a port to metastasize to other localities. As the size of the tumor increases, oxygen demand increases causing a state of hypoxia. The hypoxic state in the tumor spring forth oxygen free radicals which in turn activates vascular endothelial growth factor (VEGF) triggering the angiogenesis event. VEGF had been regarded as a heparin binding angiogenic growth factor exhibiting high specificity for endothelial cells. VEGF is responsible triggering the various steps in the angiogenesis cascade such as proliferation, migration and cell survival. The tumor regression and inhibition can be achieved by deactivating VEGF activity via neutralizing antibodies or by the introduction of dominant negative VEGF receptors into endothelial cells of tumor-associated blood vessels. Mopping up free radicals with high level of anti-oxidants can also affect transcription of the VEGF gene. This highlights the central role of VEGF in tumor angiogenesis.
Since the interdependency and a close relationship between angiogenesis, cancer growth and metastasis has well been established, much efforts have been invested into the development or the discovery of compounds with anti-angiogenic activity to target cancer and variety of other angiogenic related ailments.
The chemotherapeutic arsenals targeting neoplasm aim to cause cancer cell death via the apoptosis route. The frequency of apoptosis could contribute to cell loss in tumours and promote tumour regression. Apoptosis is accompanied by a series of morphological changes which include cell shrinkage, plasma and nuclear membrane blebbing, organelle relocalization and compaction, chromatin condensation, and production of membrane-enclosed particles containing intracellular material known as apoptotic bodies. One of the characteristic features of cancer cells is to escape from apoptosis pathways, so the induction of apoptosis is a brilliant approach to eradicate the cancerous cells without damaging the surrounding tissue. Chemotherapeutics induced apoptosis in tumor cells can occur through a number of pathways and proteins that control the cell cycle machinery including, p53, Wnt, hypoxia, NF kappa B, notch and MAP kinase pathways. Thus, in cancer therapy, the focus is on strategies that suppress tumour growth either by activating the apoptotic program in the cell or induce cell cycle arrest.
Triterpenes are a class of phytochemicals that have wide pharmacological activity. Koetjapic acid (KA) is a seco-A-ring oleanene triterpene, isolated and reported by Kaneda et al., (1992) for the first time from Sandoricum koetjape. Sandoricum koetjape is a traditional plant belonging to the family of Meliaceae. It is native to Southeast Asian countries, including Malaysia and Philippines. In Malaysia, it is locally known as santol. The tree is a medium-sized with edible fruit. In Malaysia, the aqueous extract of the bark is traditionally consumed as a tonic after giving birth, while in Indonesia it is used by folk medical practitioners to treat leucorrhoea and colic with the decoction prepared from the bark of the plant. The wood of the tree is useful for construction, being plentiful and usually easy to work and polish. It makes a good shade tree. The leaves and bark have been used medicinally as a poultice. Several parts of the plant may have anti-inflammatory effects, and some chemical extracts from santol stems have shown anti-cancer properties in vitro. Extracts from santol seeds have insecticidal properties. Cancers originating from different type of cells are, in general, very different diseases. Each cancer has characteristics that reflect its origin. Even when a cancer has metastasised and proliferated out of control, its origins can be traced back to a single, primary tumour. Therefore, it is important to develop drugs against target cells with a specified character.
Colorectal cancer, less formally known as bowel cancer, is a cancer characterized by neoplasia in the colon, rectum, or vermiform appendix. Colorectal cancers start in the lining of the bowel. If left untreated, it can grow into the muscle layers underneath, and then through the bowel wall. Most begin as a small growth on the bowel wall: a colorectal polyp or adenoma. These mushroom-shaped growths are usually benign, but some develop into cancer over time. Localized bowel cancer is usually diagnosed through colonoscopy. Colorectal cancer is the third most commonly diagnosed cancer in the world, but it is more common in developed countries. More than half of the people who die of colorectal cancer live in a developed region of the world. GLOBOCAN estimated that, in 2008, 1.23 million new cases of colorectal cancer were clinically diagnosed, and that this type of cancer killed more than 600,000 people.
The treatment of colorectal cancer depends on the stage of the cancer. When colorectal cancer is caught at early stages (with little spread), it can be curable. However, when it is detected at later stages (when distant metastases are present), it is less likely to be curable. Surgery remains the primary treatment, while chemotherapy and/or radiotherapy may be recommended depending on the individual patient's staging and other medical factors. Because colon cancer primarily affects the elderly, it can be a challenge to determine how aggressively to treat a particular patient, especially after surgery. Clinical trials suggest "otherwise fit" elderly patients fare well if they have adjuvant chemotherapy after surgery, so chronological age alone should not be a contraindication to aggressive management
In view of the above, it is advantageous to provide compounds or compositions extracted from Sandoricum koetjape that have substantial potency against colorectal cancer. SUMMARY OF THE INVENTION
The present invention provides a compound comprising the following structure, with the formula C3oH4604 and the name of seco-A-ring oleanene triterpene, also known as koetjapic acid. This compound is isolated from Sandoricum koetjape.
Figure imgf000006_0002
Figure imgf000006_0001
The above compound has anti-cancer effect and more specifically, the compound has anti-colorectal cancer effect.
The compound of the present invention is an active component identified from extracts of Sandoricum koetjape using !R, 1H and 13C NMR and mass spectrometric studies. In addition the structural elucidation and the formula was established using X-ray studies of the pure crystal of koetjapic acid.
The compound is purified and crystallised from the n-Hexane extract of the S. koetjape stem bark and the compound is found to be the major active principle in the stem bark extract of the plant.
The compound shows inhibitory activity towards human cancer cells with a higher potency towards human colorectal carcinoma (HCT 1 16) cell lines. More specifically, the compound shows anti-angiogenic activity and cytotoxic activity on human colorectal carcinoma (HCT 1 16) cell lines. The present invention provides the extract of Sandoricum koetjape against cancer growth. The cancer includes colorectal cancer.
The present invention relates to the use of extracts of Sandoricum koetjape. Extracts from leaves, branches or stems, and fruit-stems, roots and barks of the Sandoricum koetjape can be combined and the present invention discloses methods for their preparation.
The Sandoricum koetjape extracts of the present invention can be used to treat or cure cancer, particularly colorectal cancer.
The present invention also provides a pharmaceutical composition comprising an effective amount of the above-described compound and a pharmaceutical acceptable carrier(s).
The present invention further provides a method of treating a subject suffering from angiogenesis associated disease particularly but not limited to cancer, particularly but not limited to colorectal cancer. The koetjapic acid can be used as chemotherapeutic, or adjuvant to chemotherapy or as a general medicine for the treatment of cancer or angiogenesis related disorders. Herein, koetjapic acid is administered to the subject in an amount of from approximately 5 mg to about 1000 mg per kg body weight per day.
The present invention also provides a method of isolating Koetjapic acid from Sandoricum koetjape comprising the following steps: i. subjecting grounded stem bark of Sandoricum koetjape to extraction with n- hexane at around 40°C to 60°C for up to approximately 24 hours with intermittent shaking;
ii. filtering the extract and concentrating the filtered extract to dryness under reduced pressure at around 40°C to 60°C;
iii. dissolving the dried extract in a solvent mixture of methanol and acetone (1 : 1 ), and storing the dissolved reaction mixture at around -20°C (253 K) until the formation of a white precipitate; and iv. subjecting the precipitate to filtration and a series of washing with ice- chilled chloroform until the formation of koetjapic acid in the form of colourless prism-shaped crystals. It was found in the present invention that koetjapic acid exhibits the following action: inhibition of angiogenesis and inhibition of proliferation of human colorectal cancer cells.
In the present invention it was found that, koetjapic acid exhibits anti-angiogenic property by inhibiting the in vivo neovascularization in chick embryo chorioallantoic membrane (CAM) and ex vivo microvessel sprouting in rat aortic explants. Koetjapic acid inhibits angiogenesis by down regulation of VEGF signaling pathway by inhibiting the VEGF induced HUVECs proliferation, migration and differentiation, and by inhibiting the synthesis of the principal angiogenic mitogen, VEGF.
Further, the present invention provides VEGF down regulation in a composition comprising koetjapic acid as active ingredient. Thus, the composition of the present invention can be used for the treatment or prevention of diseases derived from over-expression of VEGF on the vascular system.
In the present invention it was found that, koetjapic acid exhibits anti-cancer activity by inducing apoptosis in cancer cells. It induces apoptosis by activating caspases cascade. The apoptotic efficacy of koetjapic acid is also due to its induction of nuclear fragmentation and condensation, and disruption in the mitochondrial membrane potential in cancer cells.
To detect the potential genes that contribute to angiogenesis and apoptosis in HCT 1 16 cells, the present invention also reports the early changes in the expression of genes controlling cell cycle, apoptosis and angiogenesis such as Wnt, NF-κΒ, HIF, MYC/MAX and MAPK/ERK JNK pathways were investigated.
Accordingly, the present invention provides an anti-angiogenic and anti- carcinogenic composition comprising koetjapic acid as active ingredient without other active ingredients. In the present invention carcinogenic and angiogenic related gene pathways study revealed that koetjapic acid has an extensive influence on up-regulation of N F-KB. On the other hand the compound caused significant down-regulation of five genes (MAPK/JNK, MAPK/ERK, HIF, MYC/MAX and Wnt).
Further, the present invention provides NF-κΒ up-regulation and MAPK/JNK, MAPK/ERK, HIF, MYC/MAX and Wnt down-regulation composition comprising koetjapic acid as active ingredient without other active ingredients. Thus, the composition of the present invention can be used for the treatment or prevention of diseases derived from under-expression of NF-κΒ gene and or over- expression of MAPK/JNK, MAPK/ERK, HIF, MYC/MAX and Wnt genes or pathways. Specifically, the present invention provides an anti-angiogenic and anti-tumorigenic composition comprising koetjapic acid for pharmaceutical, dietetic, health supplement and or nutraceutical uses.
Thus, the composition of the present invention can be used for the treatment or prevention of diseases derived from angiogenesis related clinical ailments.
More specifically, the present invention provides the anti-carcinogenic for colon cancer and angiogenesis-inhibitory compositions comprising koetjapic acid for pharmaceutical, dietetic, health supplement, nutraceutical, or preventive therapeutic use.
The present invention is directed to a composition comprising koetjapic acid for inhibiting angiogenesis and colorectal cancer. The composition additionally comprises at least one dose ranging from the 5 mg to 1000 mg/kg body weight. The composition may be a pharmaceutical composition for hyper-angiogenesis and cancer inhibition or prevention. The composition also may be a food composition for hyper-angiogenesis and cancer inhibition or prevention. The food composition may be a nutraceutical or health supplement. The composition is also used for prevention and/or treatment of at least one disease selected from the group consisting of cancer metastasis, obesity, arthritis, angioma, angiofibroma, diabetic retinopathy, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involutional macula, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis and pyogenic granuloma.
The present invention is also directed to a composition comprising koetjapic acid for down regulating the human VEGF signal pathway by inhibiting the VEGF protein synthesis. The composition additionally comprises at least one dose ranging from the 5 mg to 1000 mg/kg body weight. The composition may be a pharmaceutical composition for blocking VEGF signal pathway. The composition may also be a food composition for blocking VEGF signal pathway. The composition may further be a nutraceutical or health supplement. The composition may be used for treatment or prevention of at least one hyper vascularization related disease selected from the group consisting of metabolic disorder manifestations including rheumatoid arthritis, diabetic blindness, obesity, macular degeneration, Alzheimer syndrome and cancer metastasis, angioma, angiofibroma, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involutional macula, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis and pyogenic granuloma.
The present invention is also directed to a composition comprising koetjapic acid for inducing apoptosis through the activation of caspases cascade. The composition additionally comprises at least one dose ranging from the 5 mg to 1000 mg/kg body weight. The composition may be a pharmaceutical composition for blocking VEGF signal pathway. The composition may also be a food composition for blocking VEGF signal pathway. The composition may further be a nutraceutical or health supplement. The composition may be used for treatment or prevention of at least one hyper vascularization related disease selected from the group consisting of metabolic disorder manifestations including rheumatoid arthritis, diabetic blindness, obesity, macular degeneration, Alzheimer syndrome and cancer metastasis, angioma, angiofibroma, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involutional macula, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis and pyogenic granuloma. The present invention is also directed to a composition comprising koetjapic acid for the induction of nuclear fragmentation and condensation, and disruption of the mitochondrial membrane potential in cancer cells. The composition additionally comprises at least one dose ranging from approximately 5 mg to 1000 mg/kg body weight. The composition may be a pharmaceutical composition for blocking VEGF signal pathway. The composition may also be a food composition for blocking VEGF signal pathway. The composition may further be a nutraceutical or health supplement. The composition may be used for treatment or prevention of at least one hyper vascularization related disease selected from the group consisting of metabolic disorder manifestations including rheumatoid arthritis, diabetic blindness, obesity, macular degeneration, Alzheimer syndrome and cancer metastasis, angioma, angiofibroma, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involutional macula, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis and pyogenic granuloma.
The present invention is also directed to a composition comprising koetjapic acid for the over-expression of NF- Β gene and or under-expression of MAP /JNK, MAPK/ERK, HIF, YC/MAX and Wnt genes or pathways. The composition additionally comprises at least one dose ranging from the 5 mg to 1000 mg/kg body weight. The composition may be a pharmaceutical composition for blocking VEGF signal pathway. The composition may also be a food composition for blocking VEGF signal pathway. The composition may further be a nutraceutical or health supplement. The composition may be used for treatment or prevention of at least one hyper vascularization related disease selected from the group consisting of metabolic disorder manifestations including rheumatoid arthritis, diabetic blindness, obesity, macular degeneration, Alzheimer syndrome and cancer metastasis, angioma, angiofibroma, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involutional macula, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis and pyogenic granuloma.
The present invention is also directed to a composition comprising koetjapic acid for apoptotically suppress or reduce the colorectal cancer. The composition additionally comprises at least one dose ranging from the 5 mg to 1000 mg/kg body weight. The composition additionally comprises other tumorigenic human cell lines such as, MCF-7, MDA-MB231 , HepG2, COL205, CAC02, HT-29, SW480 and SW620. In addition, the composition may be a pharmaceutical composition for antitumor activity along with classical chemotherapeutic such as 5-fluro uracil against colorectal cancer. Generally the composition may also be a pharmaceutical composition for antitumor activity along with any chemotherapeutic agent against any kind of tumorigenic cancer. The composition may also be a food composition for anti-angiogenic and chemotherapeutic or chemopreventive use. The composition may further be a nutraceutical or health supplement. The composition may be used for treatment of at least one hyper-angiogenic disease selected from the group consisting of tumorigenic cancer, diabetic retinopathy, Alzheimer syndrome, rheumatoid arthritis, obesity, macular degeneration, angioma, angiofibroma, involutional macula, pterygium, retrolental fibroplasias, and pyogenic granuloma.
The present invention is also directed to a composition comprising koetjapic acid for anti-angiogencially and or apoptotically suppress or reduce the tumorigenic cancer. The composition additionally comprises for the treatment of the abnormality caused by at least one gene oncogene or the genes directly or indirectly associated with angiogenesis or cancer. The composition additionally comprises at least one angiogenesis or cancer related genes such as NF- B, MAPK/JNK, MAPK/ERK, HIF, MYC/ AX and Wnt. In addition, the composition may be a pharmaceutical composition for anti-angiogenic or antitumor activities along with any classical chemotherapeutics against cancer. The composition may also be a food composition for anti-angiogenic and chemotherapeutic or chemopreventive use. The composition may further be a nutraceutical or health supplement. The composition may be used for treatment of at least one hyper- angiogenic disease selected from the group consisting of tumorigenic cancer, diabetic retinopathy, Alzheimer syndrome, rheumatoid arthritis, obesity, macular degeneration, angioma, angiofibroma, involutional macula, pterygium, retrolental fibroplasias, and pyogenic granuloma.
In the present application, "a" and "an" are used to refer to both single and a plurality of objects. As used herein, "koetjapic acid" refers to a seco-A-ring oleanene triterpene, which can be systematically characterized as "(3R,4a 4bS,7S,8S,10bS,12aS)-7-(2- carboxyethyl)-3,4b,7,10b,12a-pentamethyl-8-(prop-1-en-2-yl)- 1 ,2,3,4,4a,4b,5,6,7,8,9,10,10b,1 1 ,12,12a-hexadecahydrochrysene-3-carboxylic acid" or it can also be systematically named as "3-((1 S,2S,4BS,6Ar,10bS)- 1 ,4b,6a,9,9,10b,-hexamethyl-2-(prop-1-en-2-yl)-
1 ,2,3,4,5,6,6a,7,8,9, 10,10a,10b, 1 1 ,12-hexadecahydrochrysen-1 -yl)propanoic acid" but without limitation to the other seco-A-ring oleanene triterpenes and the seco-A- ring oleanene triterpene derivatives which contain at least one seco-A-ring oleanene chemical skeleton or its basic nucleus that can be identified as a seco-A- ring oleanene terpene or its derivative.
Typically, koetjapic acid can be prepared synthetically or can be isolated from plants. Optionally, koetjapic acid can be prepared either by de novo synthesis or by semi-synthetica!iy modifying the basic terpenes. Koetjapic acid may be used in solid or liquid form, or it may be mixed with other liquid or solid chemicals. Alternatively, koetjapic acid may be obtained commercially in its native or modified form. In further detail and/or alternatively, "koetjapic acid" refers to a seco-A-ring oleanene triterpene and the seco-A-ring oleanene triterpene derivatives which contain at least one seco-A-ring oleanene triterpene chemical skeleton or its basic nucleus in its chemical structure which can be identified as a seco-A-ring oleanene terpene or its derivative. But it is understood that not all of the seco-A-ring oleanene triterpenes necessarily show the desired effect of anti-angiogenesis or anti-carcinogenesis.
As used herein, "angiogenesis" is meant the growth of a new blood vessel in which the proliferation and/or migration of an endothelial cell is a key step. By "inhibiting angiogenesis" or "anti-angiogenesis" is meant the inhibition of any of the steps of the process of angiogenesis that includes, without limitation, proliferation, multiplication and/or migration of endothelial cells, and it includes all aspects which cause down regulation or obstruction of the entire VEGF signal pathway. As used herein, "angiogenesis related disease" refers to those diseases that are caused by the hypervasculogenesis i.e., excessive blood vessels formation.
As used herein, "cytotoxicity" refers to either all or at least any one of the toxicity affects all cancer cell lines.
As used herein, "apoptosis" refers to either all or at least any one of the changes which leads to the death of cell. As used herein, "gene" refers to either all or at least any one of the oncogenes or the angiogenesis related genes.
As used herein, administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
For the genetic modulatory effect of koetjapic acid, the present invention reported the composition comprising the modulation of NF-κΒ, MAPK/JNK, MAPK/ERK, HIF, MYC/MAX and Wnt genes by koetjapic acid. As such, other various angiogenesis and or cancer related genes can be involved. But it is understood that not all of the angiogenesis and or cancer related genes may be necessarily involved in the regulation for the desired effect of anti-angiogenesis or anti- carcinogenesis. It will be readily apparent that all of the above compositions in their alternate forms may be used alone or in combination to provide an anti-angiogenic or anti- tumorigenic herbal product or synthetic or semi-synthetic medicine, which when given to a patient, results in the preventive or therapeutic effect against anti-tumor or angiogenesis-related aliments.
Depending on the specific clinical status of the disease, administration can be made via any accepted systemic delivery system, for example, via oral route or parenteral route such as intravenous, intramuscular, intradermal, subcutaneous or percutaneous route, or vaginal, ocular or nasal route, in solid, semi-solid or liquid dosage forms, such as for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, cream, gel, implant, patch, pessary, aerosols, collyrium, emulsions or the like, preferably in unit dosage forms suitable for easy administration of fixed dosages. The pharmaceutical compositions will include a conventional carrier or vehicle and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, and so on. In the invention, the carrier for koetjapic acid composition may preferably include, a base which is nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. If a vegetable soup or bouillon base is desired to be used as a base for the present composition, it can be readily seen that any vegetable soup or bouillon base can be used, so long as the anti-angiogenic and anticancer effects of koetjapic acid composition is maintained. if it is desired that the base be made from extracts of berries or fruits, then it is understood that any berry or fruit base may be used so long as its use does not interfere with the anti-angiogenic and anticancer effectiveness of the koetjapic acid composition. If the inventive composition is desired to be placed into soya or any other edible bean milk, it is understood that such a drink will need to be refrigerated to prevent toxic effects. It is further understood that the inventive composition may be placed, mixed, added to or combined with any other nutritional supplement so long as the anti-angiogenic and anticancer effects of the koetjapic acid composition is maintained.
If desired, the pharmaceutical composition to be administered may also contain minor amounts of heavy metals and non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like.
The amount of the koetjapic acid in a formulation can vary within the full range employed by those skilled in the art, e.g., from about 0.01 weight percent (wt %) to about 99.99 wt % of the medicine based on the total formulation and about 0.01 wt % to 99.99 wt % excipient. The preferred mode of administration, for the conditions mentioned above, is oral administration using a convenient daily dosage regimen, which can be adjusted according to the degree of the complaint. For said oral administration, a pharmaceutically acceptable, non-toxic composition is formed by the incorporation of the herbal composition in any of the currently used excipients, such as, for example, pharmaceutical grades of dextrin, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Such compositions take the form of powder, solutions, suspensions, tablets, effervescence tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may contain between 0.01 wt % and 99.99 wt % of the active compound according to this invention.
In one embodiment, the compositions will have the form of a sugar coated pill or tablet and thus they will contain, along with the active ingredient, a diluent such as lactose, sucrose, dica!cium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such as starch, polyvinylpyrrolidone, acacia gum, gelatin, cellulose and derivatives thereof, and the like. The tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active composition may be incorporated into sustained-release preparations and formulations. It is understood that by "pharmaceutical composition" or "herbal composition", it is meant that the herbal composition is formulated into a substance that is to be administered purposefully for treating or preventing anti-tumor and angiogenesis related diseases in an individual. However, it is understood that the koetjapic acid composition per se will not have a toxic effect.
As used herein, "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, experimental laboratory animals, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, and so on. Preferably, the mammal is human.
As used herein, "nutraceutical" is a combination of "nutritional" and "pharmaceutical" and refers to health supplement or food component that acts as medicines. Nutraceuticals or "functional foods" are a crude or refined specific food source that allows concentrated food therapy in a specific area of nutrition. These foods assist in the prevention or treatment of disease. Nutraceuticals may further refer to natural products that are used to supplement the diet by increasing the total dietary intake of important nutrients. Typically, nutraceuticals derived from botanical extracts or preparations such as koetjapic acid in its pure or crude form, powder or extract, and may used in the form of for example beverages made with herbal or synthetic products and other ingredients.
As used herein, "subject" is a vertebrate, preferably a mammal, more preferably a human.
As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Palliating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or the time course of the progression is slowed or lengthened, as compared to a situation without treatment. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the chemical structure of koetjapic acid;
Figure 2 depicts the iR Spectra of koetjapic acid;
Figure 3 shows the 1H NMR Spectra of koetjapic acid;
Figure 4 shows koetjapic acid inhibition of proliferation of four isogenic human tumor cell lines (HCT 116, MCF7, MDA-MB-231 , and Hep G2) and normal HUVECs in dose dependent manner. The cells exposed to koetjapic acid for 48 hours. Values are means of three experiments. Error bars equal ±SD;
Figure 5 illustrates mean clonogenic cell survival of HCT 1 16 cells treated with: (-) 1 % ethanol as a negative control, (+) betulinic acid 20 pg/rnl as a positive control, and indicated concentrations of KA. Data pooled from two experiments with error bars representing the standard deviation of means. Plating efficiency was 38.1 ± 4.6%. ***P < 0.001 ;
Figure 6 shows koetjapic acid inducing activation of caspases. Caspase 3/7, 8 and 9 activity was measured using the Caspase-Glo 3/7, 8, and 9 kit. Fold induction was calculated by dividing the values of koetjapic acid-treated samples to the readings of 0.1 % ethanol (Control). Cells treated for 3 hours. (A) Dose dependent induction of down stream caspases -3 and -7 activities. (B) Induction of upstream caspases -8 and -9 activities by koetjapic acid. Induction of caspase-9 activity is more significant than caspase-8 *P > 0.05, **P > 0.01 and ***P > 0.001 ;
Figure 7 shows morphology of apoptotic HCT 1 16 cells. Hoechst 33342-stained nucleus after 6 hours treatment with vehicle (A) 1 % ethanol, (B) 30 pg/ml, (C) 25 g/ml, (D) 20 pg/ml. The treated cells demonstrate typical apoptotic morphology (arrows): condensation of the nuclear material followed by formation of apoptotic bodies (40X). (E) Apoptotic index after treatment was calculated by dividing the number of positive-staining HCT 1 16 nuclei by the total number of HCT 1 16 nuclei after treatment with indicated concentration at indicated time and multiplying that value by 100. Error bars equal ±SD (n = 8). *P > 0.05, **P > 0.01 and > 0.001 ;
Figure 8 shows koetjapic acid inducing DNA fragmentation. HCT 1 1 16 cells were treated with 0.1 % ethanol (negative control) betulinic acid at 20 μς/ιηΙ (positive control) and the indicated concentrations of koetjapic acid for 24 hours. Both floating and adherent cells were collected, DNA was extracted and electrophoresed on a 1 .2 % agarose gel were stained with ethidium bromide and photographed;
Figure 9 shows koetjapic acid decreasing mitochondrial membrane potential as a result rhodamine 123 uptake increases extensively in treated cells. (A) Cells treated with 1 % ethanol for, (B) 20 pg/ml of koetjapic acid for 3 hours and (C) 20 pg/ml of koetjapic acid for 6 hours. After treatment, ceils were fixed by 4% paraformaldehyde and stained with rhodamine 123 (5 pg/ml) for 30 minutes. (20X);
Figure 10 shows signalling pathways influenced by koetjapic acid. Significant decrease in cancer reporter gene activities was noted for pro-proliferative pathways including Wnt, c-Myc, hypoxia, MAPK/ERC and MAPK /JNK, and significant up-regulation of NF-κΒ pathway can be seen in koetjapic acid (25 pg/mi). No significant changes detected in Notch, P53, TGF& and pRb-E2F. Error bars indicate standard deviations from mean. *P > 0.05, **P > 0.01 and ***P > 0.001 ;
Figure 1 1 shows inhibitory effect of koetjapic acid on micro-vessels formation in rat aortic rings. (A) Explants treated with 1 % ethanol as a control (B) 10 pg/ml (C) 40 pg/ml and (D) 100 pg/ml suramin as a positive control (4X). (E) The Dose response relationship of koetjapic acid on rat aorta assay. Data was represented as mean ± SD (n=12). *P > 0.05 and ***P > 0.001 ;
Figure 12 shows koetjapic acid inhibition of endothelial cell migration in a wound healing assay. A scratch is created and then the cells were treated with 1 % ethanol or 10 and 20 pg/ml of koetjapic acid. (A) At 0, 12 and 18 hours, pictures of the wounds were captured (4X) and (B) the distances between edges of the wounds calculated. Data was represented as mean ± SD (n=12). *P > 0.05 and ***P > 0.001 ; Figure 13 shows koetjapic acid inhibition of matrigel tube formation in endothelial cells. (A) Cells treated with 1 % ethanol (B) koetjapic acid (10 g/ml) (C) koetjapic acid (40 pg/ml) (D) Suramin (100 pg/ml). (E) Dose response relationship on tube formation assay. Data was represented as mean ± SD (n=12). ***P > 0.001 ; and Figure 14 shows koetjapic acid inhibition on neovascularization in chorioallantoic membrane of chick embryo: (A) Control and (B) 100 g/ml koetjapic acid treated.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to treatment for various diseases that are related to - angiogenesis and colorectal cancer. In this way, the inventive therapeutic composition may be administered to human patients who are either suffering from, or prone to suffer from the disease by providing compositions that inhibit angiogenesis and colorectal cancer. In particular, the disease is associated with a wide variety of metabolic disorder manifestations including rheumatoid arthritis, diabetic retinopathy, obesity, Alzheimer syndrome and cancer, cardiovascular diseases such as angioma, angiofibroma, vascular deformity, synechia and edemic sclerosis; and opthalmological diseases such as neovascularization after cornea implantation, neovascular glaucoma, angiogenic corneal disease, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, and granular conjunctivitis and other chronic inflammatory diseases.
In another embodiment, the present invention relates to treating various diseases that are characterized by excessive angiogenesis, which include but are not limited to, cancer, atherosclerosis, rheumatoid arthritis, endometriosis, ocular disease or obesity.
The present invention provides the method and composition comprising the isolation and crystallization of koetjapic acid from n-Henxane extract of Sandoricum koetjape stem bark. More specifically, provides the method and composition comprising the isolation and crystallization of koetjapic acid from at least one part of the plant.
The formulation of therapeutic compositions is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 5 mg to about 1000 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
It was found in the present invention that koetjapic acid of this invention inhibits angiogenesis not only in VEGF-induced HUVECs proliferation, migration and tube formation assay, but also in CAM assay and rat aortic ring assay. It was also found in the present invention that koetjapic acid of this invention inhibits angiogenesis by inhibiting the VEGF signal protein synthesis.
The HUVECs proliferation, migration and tube formation of assays are the in vitro experimental methods that are closely related to in vivo efficacy, and these assays investigate the effect on the migration and differentiation of human endothelial cell forming microvascular network. While the CAM assay is an in vivo assay using fertilized eggs, angiogenesis can be quantitatively measured in mouse Matrigel model. Furthermore, an ex vivo rat aortic ring assay, is the excellent 3 dimensional angiogenesis model, it can be used to assessed the angiogenesis quantitatively by measuring the microvessel out growths from the rat aorta.
Further, it was found in the present invention that koetjapic acid inhibited VEGF signal, an important angiogenic protein synthesized and secreted by the neoplasm in order to attract new blood vessel supply towards its own direction. It was found in the present invention that koetjapic acid significantly inhibits the production of the VEGF signal protein in cells.
It is therefore clear that the composition comprising koetjapic acid of the current invention is used as an anti-angiogenic and anti-tumorigenic agent for the treatment or prevention of angiogenesis-dependent pathologies and colorectal cancer respectively, for pharmaceutical, preventive or chemotherapeutical, nutraceutical, or health supplement, dietetic use.
It is therefore also clear that the composition comprising koetjapic acid of the present invention is used as a human VEGF signal pathway-inhibitory agent, for the treatment or prevention of the disorders arise from the over expression of VEGF proteins.
Further, it was found in the present invention that koetjapic acid down-regulated MAPK/JNK/ERK and HIF pathway genes, which are the essential pro-angiogenic genes stimulates the initiation and progression of revascularization. It was found in the present invention that koetjapic acid significantly down-regulated MAPK/JNK/ERK and HIF pathway genes in the treated colon cells. Concomitantly, it was also found in the present invention that koetjapic acid of this invention inhibits the proliferation of human colorectal tumor ceils (HCT 1 16). Koetjapic acid inhibits the growth of colon cancer cells apoptotically by inducing the activation of entire caspases cascade. It was found that koetjapic acid causes fragmentation of cellular DNA on par with the standard cytotoxic drug betulinic acid.
Further, in the present invention the apoptotic efficacy of koetjapic acid was confirmed. It was found that koetjapic acid induces apoptosis by inducing nuclear condensation and disruption in the mitochondrial membrane potential.
Further, it was found in the present invention that koetjapic acid up-regulated NF- KB gene which encodes a protein complex that controls the transcription of DNA and cell proliferation. By up-regulating NF-κΒ gene, koetjapic acid controls the growth and proliferation of cells.
Simultaneously, it was found in the present invention that koetjapic acid down- regulated MYC/MAX and Wnt genes which are pro-proliferative genes responsible for both cell proliferation. By down-regulating MYC/MAX and Wnt genes, again koetjapic acid controls the growth and proliferation of cells. S. koetjape stem used in the present invention can be purchased or cultivated in the farm. Commercially available S. koetjape stem bark or powder may also be used. S. koetjape bark n-Hexane extract of the present invention can be prepared using methods known in the art. A preferred extraction example is as follows.
In brief, 10 to 20 L of n-Hexane or the various proportional ratios of n-Hexane with chloroform, or acetone, or any other organic solvent is added to 1 kg of dried S. koetjape stem bark powder. The mixture is allowed to extract at a temperature ranging from 40 to 60°C, for a period ranging from 10 minutes to 48 hours or more. Alternatively, cold extraction procedure can also be followed at room temperature. The extraction process may be repeated 2 to 3 times with other solvents (chloroform, ethyl acetate, dichloro ethane, ketone, etc.). The resulting extract is concentrated to obtain S. koetjape extract.
As mentioned above, koetjapic acid of the present invention has inhibitory effects on neovascularization and vascular out-growth in chick embryo and rat aorta respectively. While VEGF signal pathway or MAPK/JNK/ERK and HIF genes are responsible for angiogenesis, anti-angiogenic activity of koetjapic acid is not limited to both blocking of VEGF signal pathway or down-regulating the MAPK/JNK ERK and HIF genes. That is, though VEGF or MAPK/JNK/ERK and HIF pathways are the principal factors for inducing angiogenesis, koetjapic acid can inhibit other factors of angiogenesis as well. Furthermore, the inhibitory activities of koetjapic acid on VEGF signal pathway or MAPK/JNK/ERK and genes are not limited to inhibition of angiogenesis.
In addition as described above, koetjapic acid of the present invention has potent cytotoxic effect on human colorectal cancer cell line HCT 1 16. While HCT 1 16 cell line is human colon cancer cell, anti-tumorigenic activity of koetjapic acid is not limited to HCT 1 16 cell line only. That is, though HCT 1 16 is one of the typical tumor cell lines from human colon, koetjapic can be a cytotoxic to other tumor cell lines as well. Furthermore, the inhibitory activity of koetjapic acid on proliferation of colon cell lines is not limited to inhibition of tumor. Further, in the present invention the apoptotic efficacy of koetjapic acid was confirmed. It was found that koetjapic acid induces apoptosis by inducing nuclear condensation and disruption in the mitochondrial membrane potential. As mentioned above, koetjapic acid of the present invention exhibit cytotoxicity towards colon tumor cells by inducing apoptosis. While apoptosis is responsible for cytotoxicity, but cytotoxic property of koetjapic acid is not limited to apoptosis only. That is, though apoptosis is one of the principal factors for inducing cytotoxicity, koetjapic acid can inhibit other factors of cytotoxicity as well. Furthermore, the cytotoxic property of koetjapic acid on colon tumor cells is not limited to inhibition of cell proliferation and anti-tumorigenesis.
As explained above, koetjapic acid of the present invention induces apoptosis by activating entire caspases cascade. While activation of caspases cascade is responsible for apoptosis, but activation of caspases cascade by koetjapic acid is not limited to apoptosis only. That is, though activation of caspases cascade is one of the principal factors for inducing apoptosis, koetjapic acid can activate other factors which culminate with apoptosis. Furthermore, the induction of caspases activation by koetjapic acid in cancerous cells is not limited to inhibition of ceil proliferation and anti-tumorigenesis.
As mentioned above, koetjapic acid of the present invention causes fragmentation of cellular DNA in cancerous cells. While fragmentation of cellular DNA is a characteristic of apoptosis, but DNA fragmentation caused by koetjapic acid is not limited to apoptosis only. That is, though fragmentation of cellular DNA is one of the principal factors for inducing apoptosis, koetjapic acid can induce other factors which lead to apoptosis. Furthermore, the fragmentation of cellular DNA by koetjapic acid in cancerous cells is not limited to inhibition of cell proliferation and anti-tumorigenesis.
As detailed above, koetjapic acid of the present invention causes nuclear condensation in cancerous cells. While nuclear condensation is a characteristic of apoptosis, but nuclear condensation caused by koetjapic acid is not limited to apoptosis only. That is, though nuclear condensation is one of the principal features of apoptosis, koetjapic acid can alter other factors which lead to apoptosis. Furthermore, the nuclear condensation by koetjapic acid in cancerous cells is not limited to inhibition of cell proliferation and anti-tumorigenesis.
As explained above, koetjapic acid of the present invention causes disruption in mitochondrial membrane potential in cancerous cells. While reduction of mitochondrial membrane potential is a characteristic of apoptosis, but disruption in mitochondrial membrane potential caused by koetjapic acid is not limited to apoptosis only. That is, though disruption of mitochondrial membrane potential is one of the principal features of apoptosis, koetjapic acid can affect other factors which induce apoptosis. Furthermore, the disruption of mitochondrial membrane potential by koetjapic acid in cancerous cells is not limited to inhibition of cell proliferation and anti-tumorigenesis.
As explained above, koetjapic acid of the present invention has significantly stimulated the over-expression of NF-κΒ gene. NF-κΒ gene encodes a protein complex that controls the transcription of DNA and cell proliferation. While N F-KB gene is responsible for controlling cell proliferation, anti-proliferation activity of koetjapic acid is not limited to up-regulation of NF-κΒ gene. That is, though NF- B gene is the principal factor for controlling cell proliferation, koetjapic acid can up-regulate other factors which control the cell proliferation. Furthermore, the up- regulatory activity of koetjapic acid on NF-κΒ gene is not limited to inhibition of cell proliferation and anti-tumorigenesis.
Further as reported above, koetjapic acid of the present invention has remarkably down-regulated the expression of MYC/MAX and Wnt genes responsible for both cell proliferation. While MYC/MAX and Wnt genes are responsible for controlling cell proliferation, anti-proliferation activity of koetjapic acid is not limited to down- regulation of MYC/MAX and Wnt genes. That is, though MYC/MAX and Wnt genes are the principal factors for controlling cell proliferation, koetjapic acid can down- regulate other factors which control the cell proliferation. Furthermore, the down- regulatory activity of koetjapic acid on MYC/MAX and Wnt genes are not limited to inhibition of cell proliferation and anti-tumorigenesis. The present invention provides a compound comprising the following structure, with the formula C30H46O4 Consisting of a seco-A-ring oleanene triterpene, also known as koet apic acid. This compound is isolated from Sandoricum koetjape.
Figure imgf000026_0002
Figure imgf000026_0001
Chemical Characterization of koetjapic acid
Analysis of isolated compound (koetjapic acid)
Physical state Amorphous powder
Rf value 1.3 cm (7:2:1 ethylacetate:chloroform:methanol)
Colour of spot Pink (Spraying reagent; vanillin-sulphuric acid, heated at 1 10°C) Melting point 290-292 °C (DSC)
The compound gave violet colour changing to purple in Liebermann- Burchard test for triterpenes. The molecular formula and structural elucidation of compound was established by the help of following spectroscopic data.
Spectral Characteristics of isolated compound (koetjapic acid)
IR(KBr) 3077 cm"1 , 2945 cm"1 (C-H str. in CH3 and CH2)
(see Fig . ) 70 cm"1 (C=0 str. ),
1638 cm"1 (C=C str.),
1454 cm"1 (C-H deformation in CH2/CH3),
892 cm"1 (Exocyclic CH2 with double bond)
Finger print region- 1372,1296,1264, 1227 cm"1 Ή N R (CDCI3) δ 0.9352, 0.9504, 0.9763, 1.0527, 1.2594,
(see Fig. 2) and 1.7838 (s, methyls, 18 H, 6 X CH3),
δ 1.8158 - 2.1917 (m, methylene protons, 22 H, 1 1 X CH2), δ 4.7302 (triplet) and 4.9417 (triplet), J=4.8, (2 H, for an exo-methylene group).
δ 5.3195 (s, olefinic proton)
The KBr infrared spectrum revealed the presence of carbonyl group in the structure at the region 1701 cm"1 respectively. A prominent peak at the region 1638 cm"1 corresponds to olefinic group in the structure.
The H NMR spectrum of compound contained resonances corresponding to eight methyl groups in the region range between δ 0.9352 and 1.7838 all as singlets. The methylene protons (22 H of 1 1 CH2) resonated as a multiplet at δ 1.8158 to 2.1917 and the signal exo-methylene group was observed at δ 4.7302 and 4.9417 (for 2 protons). Thus, compound was considered to be a secotriterpene.
X-Rav Crystalloqraphic study of Koetjapic acid chloroform hemisolvate
The asymmetric unit of the title compound, C3oH4604 .5CHCl3, consists of one koetjapic acid [systematic name: (3R,4a 4bS,7S,8S,10bS,12aS)-7-(2- carboxyethyl)-3,4b,7, 10b,12a-pentamethyl-8-(prop-1-en-2-yl)- 1 ,2,3,4,4a,4b,5,6,7,8,9,10,10b, 1 1 ,12,12a-hexadecahydrochrysene-3-carboxylic acid] molecule and one half-molecule of chloroform solvent, which is disordered about a twofold rotation axis. The symmetry-independent component is further disordered over two sites, with occupancies of 0.30 and 0.20. The koetjapic acid contains a fused four-ring system, A/B/C/D. The A/B, B/C and C/D junctions adopt E/trans/cis configurations, respectively. The conformation of ring A is intermediate between envelope and half-chair and ring B adopts an envelope conformation whereas rings C and D adopt chair conformations. A weak intramolecular C-Η , hydrogen bond is observed. The koetjapic acid molecules are linked into dimers by two pairs of intermolecular O-K > hydrogen bonds. The dimers are stacked along the c axis. The asymmetric unit of title koetjapic acid, consists of one koetjapic acid molecule and half a molecule of disordered chloroform solvent. The koetjapic acid contains fused four-ring system, A/B/C/D (see Fig. 1 ). The A/B, B/C and C/D junctions adopt E/trans/cis configuration, respectively. The conformation of the ring A is intermediate between envelope and half-chair [Q = 0.516 (4) A, Θ = 131.6 (3)°, φ = 45.7 (5)°]. The ring B adopts an envelope conformation [Q = 0.509 (3) A, Θ = 52.9 (3)°, φ = 177.6 (5)°] whereas ring C and D adopt chair conformations [Q = 0.561 (3) A, Θ = 155.6 (3)°, φ = 117.8 (8)°; Q = 0.491 (4) A, Θ = 168.1 (5)°, φ = 77 (2)°] (Cremer & Pople, 1975). A weak intramolecular C22— H22B-01 hydrogen bond is observed in the molecular structure.
The koetjapic acid molecules are linked into dimers by two pairs of intermolecu!ar 01— H1 -04 and 03— H3--02 hydrogen bonds (see Table 1 ). The dimers are stacked along the c axis.
The chloroform molecule is disordered over four sites in a void with twofold symmetry. In one set of symmetry-related sites, atom CI2 lies on the twofold rotation axis and the three CI sites [CM , CI2, CI1A; occupancy 0.60] are shared by two disorder components. In the other set of symmetry-related site, atoms CI3 and CI3A [occupancy 0.40] are shared by two disorder components with atoms CI4 or CI4A [occupancy 0.20] in each component. The chloroform solvent molecule lies in the cavity produced by the dimer.
Refinement:
All H atoms were positioned geometrically and refined using a riding model, with C-H = 0.93-0.98 A, Uiso(H) = 1.2 or 1.5 Ueq(C) and O-H = 0.82 A, Uiso(H) = 1.5 Ueq(O). A rotating group model was applied for the methyl groups. The chloroform molecule is disordered across a twofold rotation axis. The symmetry independent component is further disordered over two sites with occupancies of 0.30 and 0.20. All C— CI distances were restrained to be equal and Uij components of CI atoms were restrained to an approximate isotropic behaviour. The distance between atoms CI3 and CI4 at (1 -x, 2-y, z) were restrained to 2.60 (1 ) A. The highest residual electron density peak is located at 0.53 A from CI3 and the deepest hole is located at 0.68 A from CI2. 1876 Friedel pairs were used to determine the absolute configuration using the anomalous scattering of the CuKa radiation.
Table 1 depicts the basic Crystal Data obtained from the crystallographic analysis.
Table 1 : Cr stal Data
Figure imgf000029_0001
Experimental: The crystal was placed in the cold stream of an Oxford Cryosystems Cobra open-flow nitrogen cryostat (Cosier & Glazer, 1986) operating at 100.0 (1 ) K.
Geometry: All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.
Refinement: Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional f?-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > o(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Based on the evidential data from IR and 1H NMR spectra of compound suggested the presence of C-3,4-seco-olean-4,12-diene carbon skeleton in the structure and it was characterized as (3,4-seco-olean-4(23),20-diene-3,30-dioic acid which is commonly known as koetjapic acid. The structural formula of the compound is C3oH4604 (see Fig. 1 ). The complete structural characteristics were further confirmed by X-ray crystallographic studies.
Anti-proliferative efficacy of koetiapic acid Cytotoxic potency of koetjapic acid was evaluated using MTT assay on HUVEC, HCT 1 16, MCF7, MDA-MB-231 and Hep G2 cell lines. Koetjapic acid exhibited strong cytotoxicity against colorectal cancer (HCT 1 16) cell line with IC50 (concentration of test substance to achieve 50% inhibition) 18.88 pg/ml compared with other cell lines (P = 0.000). However it showed mild toxicity against other cell lines tested, although it was able to inhibit their proliferation at higher concentrations (see Fig. 4). The inhibitory effect of koetjapic acid on HCT 1 16 was comparable with the positive controls used. Table 6 depicts the IC50 values of all tested compounds on the indicated ceil lines. Cvtotstatic effect of Koetiapic acid on HCT 1 16 cells
To confirm the cytotoxic effect of KA on HCT 1 16 cells; a colony formation assay was conducted. The effect was found cytotoxic rather than cytostatic as evident by a decrease in a clonogenic survival. The PE was 38.1 ± 4.6%. At 50 and 75 Mg/ml SF was almost 0.0%. However at lower concentrations (12.5, 25 Mg/ml) the survival rates were 55.3 ± 1.6% and 41.9 ± 1.4% respectively. The calculated IC50 was 15.78 ± 1.2 pg/ml. Figure 5 depicts the survival rates of HCT 116 cells upon treatment with KA. Koetiapic acid induces apoptosis by activating caspases 3/7, 8 and 9
An attempt was made to identify whether the koetjapic acid induced inhibition of cell proliferation was due to induction of apoptosis. After three hr treatment, koetjapic acid induced the appearance of caspases activities in HCT 1 16 cells in presence of Caspase-Glo reagent. From the results it is conspicuous that, koetjapic acid activates the initiator caspases i.e., caspases 8 and 9. The activity of the initiator caspases in treated cells was about 3 fold higher than the control. Furthermore, koetjapic acid was found to be more specific for the induction of caspase 8 activity, since the induction of caspases 9 was less significant than the caspase 8. (see Fig. 6) illustrates the induction of caspases activity by koetjapic acid.
< « Morphological Modifications and nuclear condensation induced by koetjapic acid The present inventors also studied the apoptotic morphological events occurring in the nuclei after incubation of HCT 1 16 cells with koetjapic acid. No significant morphological changes were observed in nuclei incubated in the absence of koetjapic acid. At higher concentrations of koetjapic acid 30 g/ml the nuclei displayed the typical apoptotic changes in the chromatin structure appearing remarkably condensed at the nuclear periphery. Chromatin condensed progressively, in a dose dependent manner (see Fig. 7), to eventually either cluster against the nuclear periphery or to display the characteristic half-moon features observed at higher concentrations, in presence of koetjapic acid at 20 pg/ml concentration the nuclei started to shrink and the chromatin started to collapse into high density structures. The apoptotic index of koetjapic at 40 pg/ml with HCT 1 16 cells was 27.3 % and 60.3 % after 6 hr and 9 hr of incubation respectively, while at 20 g/ml the apoptotic indexes were 12 % and 30% after 6 and 9 hr respectively (see Fig. 7). DNA Fragmentation Induced by koetjapic acid
To investigate the relationship between the anti-proliferative activity and the koetjapic acid induced activity of apoptosis, DNA was extracted from the treated HCT 1 16 cells and analyzed by DNA agarose gel electrophoresis. An obvious, dose-dependent, laddering pattern was observed for the concentration range from 10 to 40 g/ml (see Fig. 8). The presence of these oligonucleosomal DNA fragments indicates that the human colorectal (HCT 1 16) carcinoma cells had undergone apoptotic cell death under the influence of koetjapic acid. However, koetjapic acid did not cause any DNA fragmentation at a concentration of 10 μg/ml. Koetjapic acid reduces Mitochondrial Potential
To investigate whether the apoptosis induced by koetjapic acid in HCT 1 16 cells involved the loss of mitochondrial integrity, the mitochondrial membrane potential in HCT 1 16 cells was evaluated by visualizing the uptake of the lipophilic cation dye rhodamine into mitochondria. The loss of mitochondrial membrane potential (ΔΨ) is a hallmark for apoptosis [32]. When the mitochondrial membrane potential decreases, the rhodamine 123 uptake by the cells increases and the florescent signal intensifies exponentially. Our results showed an obvious intensification of fluorescence in cells treated with koetjapic acid (20 g/ml) compared with control samples, which suggests a loss in mitochondrial membrane potential. Furthermore, the fluorescent intensity increased with treatment duration (see Fig. 9) the effect of koetjapic acid on mitochondrial membrane potential. Koetjapic acid modulated the gene expression in HCT 1 16
Gene expression profile has been changed greatly by the koetjapic acid (see Fig. 10). Cancer pathway study revealed that, koetjapic acid has an extensive influence on up-regulation of NF-KB (1.17 fold). On the other hand the compound caused significant down-regulation of five genes MAPK/JNK, MAPK/ERK, HIF, MYC/MAX and Wnt (0.71 , 0.56, 0.59, 0.8, 0.78 fold changes respectively). Remarkable statistical significance was obtained as shown in Fig. 10.
Koetjapic acid inhibits the sprouting of microvessels in rat aorta
In order to assess the anti-angiogenesis ability of koetjapic acid, an ex vivo rat aortic ring assay was conducted. This model was shown to be quite well correlated with in vivo events of neovascularisatio. In this assay, the rat aortic endothelium exposed to a three-dimensional matrix containing angiogenic factors switches to a microvascular phenotype generating branching networks of microvessels. As shown in Fig. 1 1 A, microvessels grew out extensively from the rat aorta in the control using 1 % ethanol as a vehicle. Whereas, koetjapic acid significantly inhibited the sprouting of rat aortic microvessels (see Figs. 11 B and 1 1 C) with IC50 16.8 g/ml. The anti-angiogenic effect on explants of rat aorta was significantly dose dependent (P<0.05). There was complete inhibition of vascularisation at the 40 pg/ml which is the IC50 for HUVECs proliferation. At a concentration of 20 g/ml koetjapic acid, which is the highest non-toxic dose on HUVECs proliferation, the observed inhibition of rat aortic microvessels was around 50 %. Suramine was used as a positive control (see Fig. 1 1 D).
Inhibitory effect of koetjapic acid on HUVECs migration
To evaluate the inhibitory effect of koetjapic acid on endothelial cell migration process, in vitro wound healing assay was conducted. This assay represents an important step in the formation of new blood vessels, and is a straightforward and economical method to study the cell migration phenomenon . A scratch wound was created on the monolayer of cells. Koetjapic acid (20 pg/ml) inhibited HUVECs migration drastically by 27.3 % after 12 hr and 23.6 % after 18 hr (P< 0.001 ). Even at lower concentration of koetjapic acid (10 pg/ml) a significant (P < 0.05) inhibitory effect of 6.9 and 10.6% inhibition after 12 and 18 hr respectively (see Fig. 12).
Inhibitory effect of koetjapic acid on tube formation in HUVECs
To characterize the anti-angiogenesis activity of koetjapic acid, we first determined whether koetjapic acid inhibited the growth factors induced tube formation of endothelial cells on Matrigel (see Fig. 13G) depicted a dose-dependent inhibition of tube formation by HUVECs. Endothelial cells formed tube-like networks (see Fig. 13A) within 6 hr, which might, in part, reflect the process of angiogenesis. At a concentration of 40 pg/ml (see Fig. 13E), the koetjapic acid absolutely abrogated endothelial tube formation by reducing the tube-like structure both in width and in length. Endothelial cells rounded up and rendered network structures incomplete and broken in the presence of koetjapic acid (see Figs. 13B-D). The activity of koetjapic acid (IC50 = 15.01 pg/ml) was comparable with that of standard drug suramin (see Fig. 13F).
In- vivo CAM assay
Vascularization in chick embryo was significantly inhibited by the 100 pg koetjapic acid. Images of two chorioallantoic membranes are shown in Fig. 14. The vasculature pattern formed by the blood vessels in CAM of control group treated with vehicle was normal. The primary, secondary and tertiary vessel with the dendritic branching pattern, which is characteristic of CAMs, was well established in control CAMs and can be seen clearly in Fig. 14A. On the other hand, the 100 g koetjapic acid treated CAM (1 mg/disc) showed the distorted architecture in vasculature, as shown in Fig. 14B.
A composition comprising koetjapic acid can also comprise more than one kind of diluent including dextrose, maltodextrin, saline, buffered saline, water, glycerol, and ethanol, but the diluent is not limited.
Formulations containing koetjapic acid may be prepared in any form. The formulation can be prepared as injectable preparation (true solution, suspension, or emulsion) and preferably in oral dosage form (tablet, effervescence tablet, capsule, soft capsule, aqueous medicine, pill, granule) and topical preparation (gel, ointment, patch, spray, solution, and the like).
The composition comprising koetjapic acid of the present invention can be administered by various routes. The route of administration includes oral, intravenous, intraperitoneal, subcutaneous, intramuscular, intra-arterial, transdermal, rectal, nasal, ocular, and topical application.
The composition comprising koetjapic acid of the present invention may be applied differently according to the diseases and route of administration, it should be understood that the amount of active ingredient has to be determined with various factors. These factors include the severity of the patient's symptoms, other coadministered drugs (e.g., chemotherapeutic agents), age, sex, body weight of the individual patient, food, dosing time, the chosen route of administration, and the ratio of the composition. A daily dose of koetjapic acid is preferable from about 5 mg to 3 g, most preferably 10 to 1000 mg. In general, 0.1 to 200 mg/kg of koetjapic acid can be administrated in a single dose at a time for 2-3 times per day.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.
Materials and Methods
Endothelial Cell Medium (ECM) supplied with endothelial cell growth supplements (ECGS) was purchased from ScienCeli, USA. M199, RPMI 1640, Dulbecco's Modified Eagle Medium (DMEM). Trypsin and heat inactivated foetal bovine serum (HIFBS) were obtained from GIBCO, UK. Human VEGF assay kit was obtained from Raybio, USA. Phosphate buffered saline (PBS), penicillin/streptomycin (PS) solution, MTT reagent, suramin, vincristine, amphotericin B, aprotinin, 6- aminocaproic acid, L-glutamine, thrombin, gentamicin, rhodamine 123 and hoechst 33342 were purchased from Sigina-Aidrich, Germany. Fibrinogen was supplied by Ca!biochem, USA and Matrigel matrix (10 mg/mL) was purchased from BD Bioscience, USA. All other chemicals used in this study were analytical grade or better. Cell lines and Culture Conditions
Human umbilical vein endothelial cell line (HUVECs) was purchased from ScienCeli, USA. HUVECs were maintained in ECM medium supplemented with 5% HIFBS, 1 % PS and 1 % ECGS. Human colorectal carcinoma (HCT 1 16), high invasive human adenocarcinama cells (MCF 7), less invasive breast ductal carcinoma cells (MDA-MB-231 ) and Hep G2 (liver cancer cell line) were purchased from American type culture collection (Rockvill, MD, USA). HCT 1 16 and cell lines were maintained in RPMI 1640 containing 10% HIFBS and 1 % PS. MCF7, MDA- MB-231 and Hep G2 were cultured in DMEM supplemented with 10% HIFBS and 1 % PS. Cells were cultured in a 5% C02-humidifed atmosphere at 37 °C.
Plant collection and authentication
The stem bark of Sandoricum koetjape was collected from the main campus of University Sains Malaysia (USM), Penang, Malaysia in the month of October 2009. The plant material was authenticated by the herbarium unit, School of Biology, USM. A voucher specimen of the plant (the leaves and the flowers) was deposited at both the herbarium unit; School of Biology and Herbal Repository, School of Pharmaceutical Sciences, USM (1 1015).
Isolation and characterization of Koetjapic acid
Koetjapic acid was isolated the from S. koetjape stem bark as described below. Briefly, 200 g of the dried powder was extracted with 000 ml of n-hexane at 40°C for 24 hours with intermittent shaking. The extract was filtered and concentrated to dryness under reduced pressure at 40°C to give 10 g of solid material. 10 g of the extract were dissolved in 50 ml, 1 :1 , methanol-acetone and was kept at -20°C. After 24 hours, a white precipitate in the amount of 500 mg was formed. The solid was filtered and washed thrice with ice-chilled chloroform. The koetjapic acid was crystallized using chloroform to yield colourless prism-shaped crystals in the amount of 400 mg. The melting point of the compound was recorded in a Toshniwal melting point apparatus. The UV spectrum was recorded on a Perkin Elmer Lambda 25 UV spectrophotometer. The IR Spectrum was recorded with KBr pellets on a Thermo nexus FT-IR spectrophotometer. The 1H NMR spectrum of the compound was taken on a Bruker AMX (300 MHz) Spectrometer using CDCI3 as solvents. X-ray analysis was studies using Bruker APEX II DUO CCD area- detector diffractometer.
Koetjapic acid was dissolved in ethanol to obtain 10 mg/ml stock solution and stored at 4°C. For drug treatment, koetjapic acid was diluted in indicated culture medium at the indicated concentrations for each experiment.
Cells Proliferation Assay Cytotoxicity of the koetjapic acid was evaluated using MTT assay against a panel of isogenic human tumor cell lines, viz. HCT 1 16, MCF7, MDA-MB-231 , Hep G2. In addition, HUVEC was also used. The assay was carried out using the method described by Mosmann et al. but with minor modification, following 48 hours of incubation. Assay plates were read using the micro-titer plate reader (Hitachi U- 2000, Japan) at 570 nm absorbance. 1 % ethanol was used as negative control. 5- flurouracil and Tamoxifen used as positives control for HCT 1 16 and MCF7 respectively.
Clonoqenic assay
In order to determine the cytotoxic or cytostatic property of koetjapic acid on HCT 1 16 cells , a clonogenic assay was carried out according to (Franken et al., 2006). In brief, 1000 cells/well within single cell suspension were plated in six well plates. After cells attachment, cells were treated with koetjapic acid (12.5, 25, 50 and 75 Mg/ml), betulinc acid 20 9/ιηΙ as a positive control or 1 % ethanol as a negative control. After 48 h of treatment, media was removed, cells were washed twice with PBS and new fresh medium was added. After 7 days the cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. The colonies of >50 cells were counted. Plating efficiency (PE) which is the ratio of the number of colonies which grown from untreated cells to the number of cells seeded was calculated using this formula:
PE = (number of colonies formed / number of cell seeded) x 100 %.
The number of colonies that arise after treatment of cells, expressed in terms of plating efficiency, is called the surviving fraction SF which was calculated according to this formula:
SF= (no. of colonies formed after treatment/ (no. of cells seeded X PE)) X 100% The results were reported as mean of percentage of survival fraction ± SD (n = 2).
Effects of koetjapic acid on caspases 3/7, 8 and 9 activities
Following treatment with koetjapic acid, HCT 1 16 cells were subjected to Caspase 3/7, 8, 9 activities measurement with Caspase-Glo assay kit (Promega, USA). In brief, after 3 hours treatment, 100 μΙ of Caspase-Glo reagent was added to each well. The plate was then incubated for 30 minutes at room temperature. The luminescence which is proportional to net caspases activities was measured in a plate-reading luminometer (HIDEX, Finland). The experiments were performed in triplicate. DNA Fragmentation Assay
HCT 1 16 cells (5 x 10 ) were treated with various concentrations of koetjapic acid (KA) for 24 hours. DNA was extracted by suspending cells in a lysis buffer provided with Wizard® SV Genomic DNA Purification kit (Promega ,USA ). DNA pellets were electrophoresed for 2 hours at 100 V in 1.2% agarose gel. The gel was stained with ethidium bromide, and the DNA fragments were visualized under ultraviolet light. Determination of nuclear condensation by Hoechst 33342 stain
Overnight incubated HCT 1 16 cells were treated with four different concentrations of koetjapic acid (KA) and analysed separately at two different time intervals (6 and 18 hours). The cells were fixed in 4% paraformaldehyde for 20 minutes before staining with Hoechst stain 33342 (1 g/ml in PBS) for 20 minutes. Nuclear condensation and cytoplasm shrinkage was examined under a fluorescent microscope (Olympus, Japan). Cells with bright condensed or fragmented nuclei were considered apoptotic. The number of cells with apoptotic morphology was counted in randomly selected fields per well. The apoptotic index was calculated as a percentage of apoptotic nuclei compared to the total number of cells and presented as the mean ± SD (n = 8).
Detection of Mitochondrial Potential Detection of the changes in mitochondrial membrane potential of HCT 1 16 cells treated with koetjapic acid (KA) was assessed by the retention of rhodamine 123. The HCT 1 16 cells were plated in 6 well plates for overnight. The cells were treated with koetjapic acid (KA) at 20 g /ml for 3 and 6 hours intervals and then fixed by 4% paraformaldehyde for 20 minutes. The rhodamine 123 was added to cells at a final concentration of 5 pg/ml and incubated for 30 minutes to stain the mitochondria. The wells were then photographed using inverted fluorescent microscope at 20X magnification power to monitor for fluorescent signals. Luciferase assay
To investigate the probable modulation of cancer pathways induced by koetjapic acid, proproliferative pathway analysis using the luciferase reporter gene assay (Cignal Finder 10; SA Biosciences, USA) was performed as per the manufactures instructions. In brief, HCT 1 16 cells were seeded into 96-well plates containing luciferase reporters to various cancer pathways along with Surefect transfection reagent, and incubated overnight for reveres transfection. Each reporter is a mixture of an inducible transcription factor responsive construct and constitutively expressing Renilla luciferase construct namely TCF/LEF, RBP-Jk, p53, SMAD2/SMAD3/SMAD4, E2F/DP1 , NF-κΒ, Myc/Max, HIF-1 , Elk-1/SRF, and AP-1 which monitor the activity of Wnt , Notch , p53 , TGF , Cell cycle , N F-KB, Myc/Max, HIF-1 , MAPK/ERK and MAPK JNK pathways respectively. Cells were treated with two concentrations of KA (25 pg/ml), 1 % ethanol in media was used as a negative control. After six hours incubation, luciferase activity was measured using the Dual Luciferase Assay system (Promega , USA). The Firefly and Renilla activities represented the experimental and normalizing reporters respectively. Reporter activity was performed in triplicate and the result was presented as a mean of a fold change ± SD for each pathway.
Ex Vivo Rat Aortic Ring Assay
This assay was carried out as described by Brown et al., with minor modifications. In brief, aortic rings were taken by cross-sectioning the thoracic aorta of 12-14 weeks old male Sprague dawley rats at 1 mm intervals after removing of periadventitial fibro adipose material and residual blood clots. Rings were positioned individually on the bottom of 48-wells plate with 300 μΙ_ serum free M199 media containing 3 mg mL"1 fibrinogen and 5 mg ml_"1 of aprotinin. 50 NIH U ml_"1 of thrombin in 0.15 M NaCI was added in each well. Immediately after embedding the vessel fragment in the fibrin gels, various concentrations of koetjapic acid (KA) dissolved in 0.3 ml of medium M 199, which was supplemented with 20% HIFBS, 0.1 % έ-aminocaproic acid, 1 % L-Glutamine, 2.5 g/ml amphotericin B, and 60 pg/ml gentamicin were added to each well. Suramin and 1 % ethanol were used as positive and negative controls respectively. The media changed at day four of the experiment .The angiogenic response was monitored by measuring the distance of blood vessels outgrowth from the primary tissue ex- plants on day five under the 4X magnification power of inverted light microscope supplied with Leica Quin computerized imaging system. Each concentration was studied in six replicates in three different experiments (n=18), the results were presented as mean percent inhibition to the negative control ± Standard Deviation (SD).
Migration Assay The assay was carried out as follows: Briefly, HUVECs were plated in 6 well plates till the formation of a confluent monolayer after which a wound was created using 200 μΙ micropipette tip. The free cells were removed by washing twice with PBS. koetjapic acid (KA) was added to the cells within a fresh media containing 10 % calf serum. After 12 to 18 hours, the wounds were photographed and distances between one side of scratch and the other were measured using inverted light microscope supplied with Leica Quin computerized imaging system. 10 fields for each concentration were captured and minimum of 10 readings of distances for each field were measured. Tube formation assay
This assay was performed using endothelial cell tube formation kit (BD biosciences, USA) as per the manufacture's manual with minor modification. In Brief, the Matrigel matrix was allowed to polymerize for 30 minutes at 37°C and 5% C02. HUVECs were trypsinized and seeded (2x104 cells per well) in each well with 50 μί of ECM containing various concentrations of koetjapic acid (KA) in triplicate. The cells were imaged under an inverted florescence microscope at low magnification. The quantitative assessment of tube formation inhibition was achieved by measuring the area of formed tubes in each field using the Scion Image analysis program. The percentage of inhibition was presented as the mean ± S.D. VEGF Quantification Assay
VEGF concentration was determined using a human VEGF-165 ELiSA kit (Raybio, USA) as per the manufacturer's instructions. The minimum detectable dose of VEGF is typically less than 20 pg/ml. ln-vivo CAM assay
This assay was performed as follows: Five day-old fertilized eggs were obtained from local farm, 5 ml of albumin were removed from the eggs and the eggs incubated horizontally allowing the CAM to detach from the shell. A small window was opened in the shell and 100 pg of the extract (prepared in ethanol) was applied onto each 6 mm disc of Whattman filter paper and then the discs were allowed to dry at 45-50°C. The loaded and dried discs were inverted and placed on the CAM. The square opening was covered with adhesive tape and the embryos incubated for 24 hours. The pictures of embryos were captured using dissecting microscope.
Statistical analysis: Results were expressed as means ± S.D. The statistical differences among the results of the various groups were compared by the one way ANOVA test. This was followed by two post-hoc tests for significance including Dunnett's (comparison groups to control) or Tukey-Kramer (all other group mean comparisons) at alpha = 0.05 , 0.01 and 0.001. The statistical analysis was carried out by using SSPS edition 12.0. The inhibition concentration (IC50) values were analyzed by log regression equation.
Results i. Chemical Characterization of koetjapic acid (KA)
Chemical Characterization of koetjapic acid
Physical state Amorphous powder
Rf value 1.3 cm (7:2:1 ethylacetate:chloroform:methanol)
Colour of spot Pink (Spraying reagent; vanillin-sulphuric acid, heated at 1 10°C) Melting point 290-292 °C (DSC)
The compound gave violet colour changing to purple in Liebermann- Burchard test for triterpenes. The molecular formula and structural elucidation of compound was established by the help of following spectroscopic data.
Spectral Characteristics of isolated compound (koetjapic acid)
IR(KBr) 3077 cm"1, 2945 cm"1 (C-H str. in CH3 and CH2)
(see Fig.2) 1701 cm"1 (C=0 str.),
1638 cm"1 (C=C str.),
1454 cm"1 (C-H deformation in CH2/CH3),
892 cm"1 (Exocyclic CH2 with double bond)
Finger print region- 1372, 1296,1264, 1227 cm"1
1H NMR (CDCI3) δ 0.9352, 0.9504, 0.9763, .0527, 1 .2594,
(see Fig.3) and 1.7838 (s, methyls, 18 H, 6 X CH3),
δ 1.8158 - 2.1917 (m, methylene protons, 22 H, 1 1 X CH2), δ 4.7302 (triplet) and 4.9417 (triplet), J=4.8, (2 H, for an exo- methylene group),
δ 5.3 95 (s, olefinic proton)
The KBr infrared spectrum revealed the presence of carbonyl group in the structure at the region 1701 cm"1 respectively. A prominent peak at the region 1638 cm"1 corresponds to olefinic group in the structure.
The 1H NMR spectrum of compound contained resonances corresponding to eight methyl groups in the region range between δ 0.9352 and 1.7838 all as singlets. The methylene protons (22 H of 1 1 CH2) resonated as a multiplet at δ 1.8158 to 2.1917 and the signal exo-methylene group was observed at δ 4.7302 and 4.9417 (for 2 protons). Thus, compound was considered to be a secotriterpene. X-Rav Crvstalloqraphic study of Koetjapic acid chloroform hemisolvate
The asymmetric unit of the title compound, C3oH4604-0.5CHCI3, consists of one koetjapic acid [systematic name: (3R,4aR,4bS,7S,8S,10bS,12aS)-7-(2- carboxyethyl)-3,4b,7, 10b, 12a-pentamethyl-8-(prop-1 -en-2-yl)-
1 ,2,3,4,4a,4b,5,6,7,8,9,10,10b,11 ,12,12a-hexadecahydrochrysene-3-carboxylic acid] molecule and one half-molecule of chloroform solvent, which is disordered about a twofold rotation axis. The symmetry-independent component is further disordered over two sites, with occupancies of 0.30 and 0.20. The koetjapic acid contains a fused four-ring system, A/B/C/D. The A/B, B/C and C/D junctions adopt E/trans/cis configurations, respectively. The conformation of ring A is intermediate between envelope and half-chair and ring β adopts an envelope conformation whereas rings C and D adopt chair conformations. A weak intramolecular C-H.. hydrogen bond is observed. The koetjapic acid molecules are linked into dimers by two pairs of intermolecular O-H.. hydrogen bonds. The dimers are stacked along the c axis.
The asymmetric unit of title koetjapic acid, consists of one koetjapic acid molecule and half a molecule of disordered chloroform solvent. The koetjapic acid contains fused four-ring system, A B/C/D. The A/B, B/C and C/D junctions adopt E/trans/cis configuration, respectively. The conformation of the ring A is intermediate between envelope and half-chair [Q = 0.516 (4) A, Θ = 131.6 (3)°, φ = 45.7 (5)°]. The ring B adopts an envelope conformation [Q = 0.509 (3) A, Θ = 52.9 (3)°, φ = 177.6 (5)°] whereas ring C and D adopt chair conformations [Q = 0.561 (3) A, Θ = 155.6 (3)°, φ = 1 17.8 (8)°; Q = 0.491 (4) A, Θ = 168.1 (5)°, φ = 77 (2)°] (Cremer & Pople, 1975). A weak intramolecular C22— Η22Β-Ό1 hydrogen bond is observed in the molecular structure.
The koetjapic acid molecules are linked into dimers by two pairs of intermolecular 01— H1 -04 and 03— H3-02 hydrogen bonds (Table 1 ). The dimers are stacked along the c axis.
The chloroform molecule is disordered over four sites in a void with twofold symmetry. In one set of symmetry-related sites, atom CI2 lies on the twofold rotation axis and the three CI sites [CM , CI2, CM A; occupancy 0.60] are shared by two disorder components. In the other set of symmetry-related site, atoms CI3 and CI3A [occupancy 0.40] are shared by two disorder components with atoms CI4 or CI4A [occupancy 0.20] in each component. The chloroform solvent molecule lies in the cavity produced by the dimer.
Refinement:
All H atoms were positioned geometrically and refined using a riding model, with C-H = 0.93-0.98 A, Uiso(H) = 1 .2 or 1.5 Ueq(C) and O-H = 0.82 A, Uiso(H) = 1.5 Ueq(O). A rotating group model was applied for the methyl groups. The chloroform molecule is disordered across a twofold rotation axis. The symmetry independent component is further disordered over two sites with occupancies of 0.30 and 0.20. All C— CI distances were restrained to be equal and Uij components of CI atoms were restrained to an approximate isotropic behaviour. The distance between atoms CI3 and CI4 at (1 -x, 2-y, z) were restrained to 2.60 (1 ) A. The highest residual electron density peak is located at 0.53 A from CI3 and the deepest hole is located at 0.68 A from CI2. 1876 Friedel pairs were used to determine the absolute configuration using the anomalous scattering of the CuKa radiation. Table 1 on page 27 depicts the basic Crystal Data obtained from the crystallographic analysis.
Table 2 depicts the fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (A2)
Table 2
X y Z / *IU Occ. (<1 )
01 0.2093 (2) 0.96882 (8) 0.3593 (5) 0.0439 (8)
H1 0.2417 0.9868 0.4147 0.066*
02 0.1261 (2) 0.96828 (8) 0.6601 (5) 0.0436 (8)
03 0.7713 (3) 0.9691 1 (9) 0.8069 (6) 0.0514 (9)
H3 0.8000 0.9887 0.7590 0.077*
04 0.6996 (5) 0.96705 (14) 0.4969 (7) 0.106 (2) C1 0.5175 (3) 0.84482 (1 1 ) 0.6074 (6) 0.0304 (8)
C2 0.4878 (3) 0.86704 (12) 0.4167 (6) 0.0329 (9)
H2A 0.5314 0.8577 0.3056 0.039*
H2B 0.5037 0.8949 0.4378 0.039*
C3 0.3751 (3) 0.86394 (12) 0.3479 (6) 0.0328 (9)
H3A 0.3678 0.8412 ' 0.2578 0.039*
H3B 0.3572 0.8875 0.2700 0.039*
C4 0.2988 (3) 0.85964 (10) 0.5262 (6) 0.0262 (8)
C5 0.1868 (3) 0.85387 (10) 0.4384 (6) 0.0264 (8)
H5A 0.1946 0.8317 0.3427 0.032*
C6 0.1433 (3) 0.88781 (10) 0.3023 (6) 0.0292 (8)
H6A 0.2020 0.9006 0.2369 0.035*
H6B 0.1027 0.8755 0.1944 0.035*
C7 0.0758 (3) 0.92092 (1 1 ) 0.3973 (7) 0.0339 (9)
C8 0.0003 (3) 0.90414 (12) 0.5567 (7) 0.0391 (10)
H8A -0.0273 0.9259 0.6374 0.047*
H8B -0.0574 0.8918 0.4859 0.047*
C9 0.0491 (3) 0.87389 (12) 0.7005 (7) 0.0354 (9)
H9A -0.0044 0.8636 0.7905 0.042*
H9B 0.1000 0.8873 0.7851 0.042*
C10 0.1020 (3) 0.83885 (1 1 ) 0.5927 (6) 0.0300 (9)
C1 1 0.1466 (3) 0.81017 (12) 0.7559 (6) 0.0356 (9)
H1 1A 0.1008 0.8107 0.8737 0.043*
H1 1 B 0.1444 0.7835 0.7001 0.043*
C12 0.2567 (3) 0.81809 (12) 0.8308 (6) 0.0325 (9)
H12A 0.2788 0.7966 0.9190 0.039*
H12B 0.2578 0.8424 0.9103 0.039*
C13 0.3322 (3) 0.82188 (10) 0.6488 (5) 0.0252 (8)
C14 0.4463 (3) 0.82387 (10) 0.7164 (5) 0.0254 (8)
C15 0.4753 (3) 0.79887 (1 1 ) 0.8945 (6) 0.0303 (8) H15A 0.4413 0.7734 0.8803 0.036*
H15B 0.4484 0.8113 1.0176 0.036*
C16 0.5911 (3) 0.79172(12) 0.9237 (6) 0.0346 (9)
H16A 0.6142 0.7710 0.8312 0.042*
H16B 0.6043 0.7830 1.0628 0.042*
C17 0.6514(3) 0.82981 (11) 0.8812(6) 0.0304 (8)
H17A 0.6197 0.8505 0.9658 0.036*
C18 0.6348 (3) 0.84244(11) 0.6529 (6) 0.0270 (8)
C19 0.6891 (3) 0.88239(12) 0.6111 (6) 0.0335 (9)
H19A 0.7628 0.8790 0.6352 0.040*
H19B 0.6803 0.8888 0.4673 0.040*
C20 0.6517(4) 0.91792 (12) 0.7379 (7) 0.0440 (11)
H20A 0.5779 0.9218 0.7178 0.053*
H20B 0.6640 0.9130 0.8823 0.053*
C21 0.7097 (5) 0.95397(14) 0.6711 (8) 0.0539 (13)
C22 0.3104(3) 0.89708(11) 0.6604 (6) 0.0325 (9)
H22A 0.2509 0.8996 0.7478 0.049*
H22B 0.3157 0.9200 0.5742 0.049*
H22C 0.3718 0.8948 0.7427 0.049*
C23 0.1407 (3) 0.95455(11) 0.4877 (7) 0.0338 (9)
C24 0.0136 (4) 0.94111 (12) 0.2222 (8) 0.0453(11)
H24A 0.0608 0.9509 0.1205 0.068*
H24B -0.0260 0.9627 0.2770 0.068*
H24C -0.0324 0.9221 0.1610 0.068*
C25 0.0193(3) 0.81565(11) 0.4729 (7) 0.0378 (9)
H25A -0.0317 0.8055 0.5663 0.057*
H25B 0.0515 0.7940 0.4017 0.057*
H25C -0.0136 0.8329 0.3758 0.057*
C26 0.3264 (3) 0.78339(11) 0.5175 (6) 0.0326 (9)
H26A 0.3786 0.7842 0.4128 0.049* H26B 0.2592 0.7815 0.4551 0.049*
H26C 0.3378 0.7607 0.6036 0.049*
C27 0.7649 (3) 0.82688 (13) 0.9473 (6) 0.0357(9)
C28 0.8203 (3) 0.79269 (13) 0.9245 (7) 0.0440(11)
H28A 0.8880 0.7912 0.9730 0.066*
H28B 0.7904 0.7709 0.8604 0.066*
C29 0.8118(3) 0.86138(13) 1.0484 (6) 0.0407(10)
H29A 0.8764 0.8538 1.1099 0.061*
H29B 0.7657 0.8711 1.1523 0.061*
H29C 0.8240 0.8818 0.9491 0.061*
C30 0.6836 (3) 0.81256 (12) 0.5030 (6) 0.0361 (9)
H30A 0.6690 0.8205 0.3649 0.054*
H30B 0.6549 0.7867 0.5271 0.054*
H30C 0.7573 0.8118 0.5236 0.054*
C31 0.5335(9) 0.9947 (3) 0.0839(17) 0.087 (5) 0.50
H31A 0.6041 0.9856 0.0890 0.104* 0.299 (3)
H31B 0.5871 0.9780 0.1384 0.104* 0.201 (3)
C11 0.4660 (3) 0.95921 (9) 0.1615 (9) 0.1061 (18) 0.597 (7)
CI2 0.5000 1.0000 -0.2013(6) 0.127 (3) 0.597 (7)
CI3 0.4269 (6) 0.9672 (3) -0.0147(14) 0.153 (4) 0.403 (7)
CI4 0.4584 (9) 1.0233 (3) 0.2864(15) 0.107 (4) 0.201 (3)
Table 3 shows the atomic displacement parameters (A2)
Table 3
U22 L/33 12 L/3 L/23
0.0454 0.0302 0.0561 -0.0102 -0.0052
01 0.0042(15)
(17) (14) (18) (13) (14)
0.0436 0.0284 -0.0053 -0.0103
02 0.059 (2) 0.0067 (15)
(17) (14) (12) (14)
03 0.0545 0.0332 0.067 (2) -0.0172 -0.0117 0.0117(15) (19) (15) (14) (17)
04 0.166 (5) 0.075 (3) 0.076 (3) -0.080 (3) -0.044 (3) 0.034 (2)
0.0276 0.0302 -0.0056 -0.0014
C1 0.033 (2) 0.0033 (15) (19) (19) (16) (16)
0.0252
C2 0.030 (2) 0.044 (2) 0.0015 (17) 0.0039 (16) 0.0049 (16)
(19) >
-0.0023 -0.0003
C3 0.033 (2) 0.036 (2) 0.030 (2) 0.0074 (16)
(16) (16)
0.0269 0.0229 0.0287 -0.0009 -0.0021
C4 0.0005 (15)
(18) (17) (18) (14) (14)
0.0267 0.0226 0.0298 -0.0008 -0.0016 -0.0012
C5
(18) (17) (19) (14) (15) (14)
0.0297 0.0253 -0.0020 -0.0062 -0.0020
C6 0.033 (2)
(19) (18) (15) (16) (15)
0.0256 0.0235 -0.0041 -0.0037
C7 0.053 (2) 0.0005 (15)
(19) (18) (19) (17)
0.0284 -0.0028 -0.0086
C8 0.030 (2) 0.059 (3) 0.0018 (19)
(19) (17) (18)
-0.0044 -0.0050
C9 0.027 (2) 0.036 (2) 0.043 (2) 0.0065 (17)
(16) (18)
0.0264 0.0276 -0.0038 -0.0006
C10 0.036 (2) 0.0027 (16)
(18) (19) (15) (16)
-0.0039
C1 1 0.030 (2) 0.036 (2) 0.041 (2) 0.0063 (18) 0.0087 (17)
(17)
C12 0.031 (2) 0.036 (2) 0.031 (2) 0.0012 (16) 0.0034 (16) 0.0055 (17)
0.0256 0.0250 0.0250 -0.0030
C13 0.0028 (14) 0.0002 (14) (18) (17) (17) (14)
0.0205 0.0246 -0.0027 -0.0020 -0.0041
C14 0.031 (2)
(17) (17) (14) (14) (14)
0.0255 -0.0047
C15 0.034 (2) 0.031 (2) 0.0044 (17) 0.0018 (15)
(18) (16)
C16 0.038 (2) 0.033 (2) 0.033 (2) -0.0072 -0.0078 0.0074 (16) (18) (17)
0.0301 0.0321 0.0288 -0.0076 -0.0003
C17 0.0018 (15) (19) (19) (18) (16) (16)
0.0269 0.0263 0.0277 -0.0041
C18 0.0005 (15) 0.0024 (14) (19) (18) (18) (15)
-0.0099 «
C19 0.030 (2) 0.036 (2) 0.035 (2) 0.0028 (17) 0.0069 (16)
(16)
-0.01 12 -0.0015
C20 0.048 (3) 0.032 (2) 0.052 (3) 0.004 (2)
(19) (19)
C21 0.075 (4) 0.033 (2) 0.054 (3) -0.014 (2) -0.013 (3) 0.005 (2)
0.0271 -0.0031 -0.0067 -0.0032
C22 0.032 (2) 0.038 (2)
(19) (16) (17) (16)
0.0221 -0.0005 -0.0031
C23 0.030 (2) 0.050 (2) 0.0003 (15)
(18) (19) (18)
C24 0.037 (2) 0.031 (2) 0.067 (3) 0.0004 (18) -0.012 (2) 0.003 (2)
-0.0047 ' -0.0031 -0.0002
C25 0.031 (2) 0.028 (2) 0.054 (2)
(17) (19) (18)
0.0250 -0.0009 -0.0048 -0.0022
C26 0.032 (2) 0.041 (2)
(18) (15) (17) (16)
0.0267 -0.0099 -0.0052
C27 0.037 (2) 0.043 (2) 0.0083 (17)
(19) (18) (16)
-0.0047
C28 0.036 (2) 0.042 (2) 0.054 (3) -0.013 (2) 0.009 (2)
(19)
-0.0166 -0.0029
C29 0.037 (2) 0.050 (3) 0.035 (2) 0.0055 (18)
(19) (18)
-0.0022 -0.0023
C30 0.036 (2) 0.040 (2) 0.032 (2) 0.0063 (17)
(17) (17)
C31 0.066 (8) 0.051 (7) 0.143 (13) 0.020 (6) -0.016 (8) -0.002 (8)
0.0606 -0.0130
CI1 0.103 (3) 0.154 (4) 0.036 (3) 0.032 (2)
(17) (16)
CI2 0.245 (7) 0.078 (3) 0.059 (2) 0.095 (4) 0.000 0.000
CI3 0.154 (6) 0.141 (6) 0.162 (7) -0.086 (5) 0.055 (5) -0.070 (6) CI4 0.151 (8) 0.077 (5) 0.094 (6) -0.008 (6) 0.001 (6) -0.038 (5)
Table 4 depicts the geometric parameters (A, °)
Table 4
01— C23 1.310 (5) C17— C27 1 ,529 (6)
01— H1 0.82 C17— C18 1.565 (5)
02— C23 1.232 (5) C17— H17A 0.98
03— C21 1.296 (6) C18— C30 1.539 (5)
03— H3 0.82 C18— C19 1.543 (5)
04— C21 1.227 (6) C19— C20 1.534 (6)
C1— C14 1.360 (5) C19— H19A 0.97
C1— C2 1 .504 (5) C19— H19B 0.97
C1— C18 1 .544 (5) C20— C21 1.492 (6)
C2— C3 1.525 (6) C20— H20A 0.97
C2— H2A 0.97 C20— H20B 0.97
C2— H2B 0.97 C22— H22A 0.96
C3— C4 1.531 (5) C22— H22B 0.96
C3— H3A 0.97 C22— H22C 0.96
C3— H3B 0.97 C24— H24A 0.96
C4— C22 1.545 (5) C24— H24B 0.96
C4— C13 1.565 (5) C24— H24C 0.96
C4— C5 1.565 (5) C25— H25A 0.96
C5— C6 1.554 (5) C25— H25B 0.96
C5— C10 1 .571 (5) C25— H25C 0.96
C5— H5A 0.98 C26— Ή26Α 0.96
C6— C7 1.546 (5) C26— H26B 0.96
C6— H6A 0.97 C26— H26C 0.96
C6— H6B 0.97 C27— C28 1.365 (6)
C7— C23 1.528 (5) C27— C29 1 .468 (6) 07— C8 1.534 (6) C28— H28A 0.93
C7— C24 1.554 (6) C28— H28B 0.93
C8— C9 1.523 (6) C29— H29A 0.96
C8— H8A 0.97 C29— H29B 0.96
C8— H8B 0.97 C29— H29C 0.96
C9— C10 1.536 (5) C30— H30A 0.96
C9— H9A 0.97 C30— H30B 0.96
C9— H9B 0.97 C30— H30C 0.96
C10— C25 1.538 (6) C31— 031' 0.94 (2)
C10— C11 1.550 (5) C31— CI4' 1.460 (13)
C11— C12 1.525 (6) C31— 013* 1.526 (10)
C11— H11A 0.97 C31— CM 1.564 (9)
C11— H11B 0.97 C31— Oil' 1.635 (9)
C12— C13 1.543 (5) C31— CI3 1.779 (11)
C12— H12A 0.97 C31— Cl4 1.903 (12)
C12— H12B 0.97 C31— CI2 1.920 (12)
C13— C14 1.538 (5) C31— H31A 0.96
C13— C26 1.558 (5) C31— H31B 0.96
C14— C15 1.484 (5) 011—031' 1.635 (9)
C15— C16 1.525 (6) CI1— H31B 1.6921
C15— H15A 0.97 012—031' 1.920 (12)
C15— H15B 0.97 013—031' 1.526 (10)
C16— C17 1.527 (5) CI4— 031' 1.460(13)
C16— H16A 0.97 CI4— CI4' 1.91 (2)
C16— H16B 0.97
C23— 01— H1 109.5 H16A— 016— H16B 108.2
C21— 03— H3 109.5 016— 017— 027 112.4 (3)
C14— C1— C2 121.4(3) 016— 017— 018 109.4(3)
C14— C1— C18 122.3(3) 027— 017— 018 114.7 (3) C2— C1— C18 115.8(3) C16— C17— H17A 106.6
C1— C2— C3 116.9(3) C27— C17— H17A 106.6
C1— C2— H2A 108.1 C 8— C17— H17A 106.6
C3— C2— H2A 108.1 C30— C18— C19 105.9(3)
C1— C2— H2B 108.1 C30— C18— C1 108.2(3)
' C3— C2— H2B 108.1 C19— C18— C1 111.4 (3)
H2A— C2— H2B 107.3 C30— C18— C17 111.8 (3)
C2— C3— C4 113.3(3) C19— C18— C 7 110.1 (3)
C2— C3— H3A 108.9 C1— C18— C17 109.3(3)
C4— C3— H3A 108.9 C20— C19— C18 116.4(3)
C2— C3— H3B 108.9 C20— C19— H19A 108.2
C4— C3— H3B 108.9 C18— C19— H19A 108.2
H3A— C3— H3B 107.7 C20— C19— H19B 108.2
C3— C4— C22 107.0(3) C18— C19— H19B 108.2
C3— C4— C13 106.8 (3) H19A— C19— H19B 107.3
C22— C4— C13 110.4(3) C21— C20— C19 108.8(4)
C3— C4— C5 109.0 (3) C21— C20— H20A 109.9
C22— C4— C5 113.5 (3) C19— C20— H20A 109.9
C13— C4— C5 109.9(3) C21— C20— H20B 109.9
C6— C5— C4 116.8 (3) C19—C20— H20B 109.9
C6— C5— C10 110.7(3) H20A— C20— H20B 108.3
C4— C5— C10 116.6(3) 04— C21—03 123.9(5)
C6— C5— H5A 103.5 04— C21— C20 120.7(5)
C4— C5— H5A 103.5 03— C21— C20 115.4(4)
C10— C5— H5A 103.5 C4— C22— H22A 109.5
C7— C6— C5 120.4(3) C4— C22— H22B 109.5
C7— C6— H6A 107.2 H22A— C22— H22B 109.5
C5— C6— H6A 107.2 C4— C22— H22C 109.5
C7— C6— H6B 107.2 H22A— C22— H22C 109.5
C5— C6— H6B 107.2 H22B— C22— H22C 109.5 H6A-— C6— H6B 106.9 02— C23— 01 123.4(4)
C23- -C7— C8 111.1 (4) 02— C23— C7 123.2(4)
C23- -C7— C6 112.5(3) 01— C23— C7 113.3(4)
C8- -C7— C6 111.3(3) C7— C24— H24A 109.5
C23- -C7— C24 104.0(3) C7— C24— H24B 109.5
C8- -C7— C24 109.5(3) H24A— C24— H24B 109.5
C6- -C7— C24 108.2 (4) C7— C24— H24C 109.5
C9- -C8— C7 113.8(3) H24A— C24— H24C 109.5
C9- -C8— H8A 108.8 H24B— C24— H24C 109.5
C7- -C8— H8A 108.8 C10— C25— H25A 109.5
C9- ■C8— H8B 108.8 C10— C25— H25B 109.5
C7- -C8— H8B 108.8 H25A— C25— H25B 109.5
H8A- — C8— H8B 107.7 C10— C25— H25C 109.5
C8- -C9— CIO 14.6 (4) H25A— C25— H25C 109?5
C8- -C9— H9A 108.6 H25B— C25— H25C 109.5
C10- — C9— H9A 108.6 C13— C26— H26A 109.5
C8- -C9— H9B 108.6 C13— C26— H26B 109.5
C10- — C9— H9B 108.6 H26A— C26— H26B 109.5
H9A- — C9— H9B 107.6 C13— C26— H26C 109.5
C9- -C10— C25 108.5 (3) H26A— C26— H26C 109.5
C9- -C10— C11 109.3(3) H26B— C26— H26C 109.5
C25- -C10— C11 106.8(3) C28— C27— C29 120.2 (4)
C9- -C10— C5 110.8(3) C28— C27— C17 121.7(4)
C25- -C10— C5 108.7(3) C29— C27— C17 118.0(4)
C11- -C10— C5 112.6(3) C27— C28— H28A 120.0
C12- -C11— C10 117.1 (3) C27— C28— H28B 120.0
C12- -C11— H11A 108.0 H28A— C28— H28B 120.0
C10- -C11— H11A 108.0 C27— C29— H29A 109.5
C12- -C11— H11B 108.0 C27— C29— H29B 109.5
C10- -C11— H11B 108.0 H29A— C29— H29B 109.5 H11A— C11— H11B 107.3 C27— C29— H29C 109.5
C11— C12— C13 110.8(3) H29A— C29— H29C 109.5
C11— C12— H12A 109.5 H29B— C29— H29C 109.5
C13— C12— H12A 109.5 C18— C30— H30A 109.5
C11— C12— H12B 109.5 C18— C30— H30B 109.5
C13— C12— H12B 109.5 H30A— C30— H30B 109.5
H12A— C12— H12B 108.1 C18— C30— H30C 109.5
C14— C13— C12 112.8 (3) H30A— C30— H30C 109.5
C14— C13— C26 103.9 (3) H30B— C30— H30C 109.5
C12— C13— C26 109.0 (3) CW1— C31— CI31 135.2 (9)
C14— C13— C4 112.0 (3) CI1— C31— cir 129.0 (8)
C12— C13— C4 106.7(3) CW1— C31— CI3 99.6 (7)
C26— C13— C4 112.5 (3) CI3'— C31— CI3 123.0 (9)
C1— CI 4— C15 122.4(3) CW— C31— CI4 67.6 (10)
C1— C14— C13 121.2 (3) CI3'— C31— CI4 92.0 (6)
C15— C14— C13 116.2 (3) C!3— C31— CI4 97.1 (6)
C14— C15— C16 115.7(3) CI1— C31— CI2 105.1 (6)
C14— C15— H15A 108.3 CM'— C31— CI2 102.3 (6)
C16— C15— H15A 108.3 CI1— C31— H31A 105.8
C14— C15— H15B 108.3 CIV— C31— H31A 106.8
C16— C15— H15B 108.3 CI2— C31— H31A 106.1
H15A— C15— H15B 107.4 C\A[— C31— H31B 50.8
C15— C16— C17 110.0(3) C\3— C31— H31B 114.2
C15— C16— H16A 109.7 CI3— C31— H31B 112.6
C17— C16— H16A 109.7 CI4— C31— H31B 114.0
C15— C16— H16B 109.7 CI2— C31— H31B 125.2
C17— C16— H16B 109.7
C14— C1— C2— C3 -1.8(6) C15— C16— C17— C18 -62.5 (4)
C18— C1— C2— C3 169.8(3) C14— C1— C18— C30 97.3 (4)
C1— C2— C3— C4 32.3 (5) C2— C1— C18— C30 -74.3 (4) C2— C3— C4— C22 60.6 (4) C14- -C1— C18— C19 -146.7 (4)
C2— C3— C4— C13 -57.6 (4) C2- ■C1— C18— C19 41.7 (5)
C2— C3— C4— C5 -176.3 (3) C14- -C1— C18— C17 -24.7 (5)
C3— C4— C5— C6 -60.2 (4) C2- -C1— C18— C17 163.7 (3)
C22— C4— C5— C6 58.9 (4) C16- -C17— C18— C30 -66.4 (4)
C13— C4— C5— C6 -176.9 (3) C27- -C17— C18— C30 61.0 (4)
C3— C4— C5— C10 165.8 (3) C16- -C17— C18— C19 176.2 (3)
C22— C4— C5— C10 -75.1 (4) C27- -C17— C18— C19 -56.5 (4)
C13— C4— C5— C10 49.1 (4) C16- -C17— C18— C1 53.4 (4)
C4—C5— C6— C7 -94.0 (4) C27- -C17— C18— C1 -179.2 (3)
C10— C5— C6— C7 42.5 (4) C30- -C18— C19— C20 178.2 (4)
C5— C6— C7— C23 85.3 (4) C1- -C18— C19— C20 60.8 (5)
C5— C6— C7— C8 -40.2 (5) C17- -C18— C19— C20 -60.7 (5)
C5— C6— C7— C24 -160.5 (3) C18- -C19— C20— C21 -177.8 (4)
C23— C7— C8— C9 -82.4 (4) C19- -C20— C21— 04 64.4 (7)
C6— C7— C8— C9 43.8 (5) C19- -C20— C21— 03 -1 13.9 (5)
C24— C7— C8— C9 163.3 (3) C8- -C7— C23— 02 -6.2 (5)
C7— C8— C9— C10 -55.0 (5) C6- -C7— C23— 02 -131.8 (4)
C8— C9— C10— C25 -63.0 (4) C24- -C7— C23— 02 1 1 1.5 (5)
C8— C9— C10— C1 1 -179.1 (3) C8- -C7— C23— 01 177.5 (3)
C8— C9— C10— C5 56.3 (4) C6- -C7— C23— 01 52.0 (5)
C6— C5— C10— C9 -47.5 (4) C24- -C7— C23— 01 -64.8 (4)
C4— C5— C10— C9 89.1 (4) C16- -C17— C27— C28 37.4 (5)
C6— C5— C10— C25 71.6 (4) C18- -C17— C27— C28 -88.4 (5)
C4— C5— C10— C25 -151.8 (3) C16- -C17— C27— C29 -139.0 (4)
C6— C5— C10— C1 1 -170.3 (3) C18- -C17— C27— C29 95.2 (4)
C4— C5— C10— C1 1 -33.6 (4) C\ - -C31— CM— C311 1 16.2 (8)
C9— C10— C1 1— C12 -88.7 (4) c\y- -C31— CI1— C311 -23 (4)
C25— C10— C1 1— C12 154.1 (4) cir- -C31— CI1— C311 48.8 (10)
C5— C10— C1 1— C12 34.9 (5) CI3- -C31— CI1— C311 -58.6 (5) C10— C11— C12— C13 -52.2 (5) CI4- -C31- -CI1--C3V 48.8 (6)
C11— C12— C13— C14 -171.7(3) CI2- -C31- -CI1- -C3V -71.3(5)
C11—C12— C13— C26 -56.9 (4) CI4j- -C31- -CI2- -C3V 90 (3)
C11— C12— C13— C4 64.9 (4) CI3'- -C31- -CI2- -C3V -95.7(11)
C3— C4— C13— C14 55.0 (4) CI1- -C31- -CI2- -C311 72.3(11)
C22—C4— C13— C14 -60.9 (4) cir- -C31- -CI2- -C31' '-64.2(9)
C5— C4— C13— C14 173.1 (3) CI3- -C31- -CI2- -C3V 62.1 (10)
C3— C4— C13— C12 178.9(3) CI4- -C31- -CI2- -C3V -18.6(7)
C22— C4— C13— C12 63.0 (4) CI4i- -C31- -CI3- -C3Y 99.5(8)
C5— C4— C13— C12 -63.0 (4) c\y- -C31- -CI3- -C3V -65.9 (11)
C3— C4— C13— C26 -61.7(4) CI1- -C31- -CI3- -C3V 103.9(6)
C22— C4— C13— C26 -177.6 (3) cir- -C31- -CI3- -C3V -6.6 (11)
C5— C4— C13— C26 56.4 (4) CI4- -C31- -CI3- -C3V 31.2(7)
C2— C1— C14— C15 174.5(3) CI2- -C31- -CI3- -C3V -90.1 (6)
C18— C1— C14— C15 3.5(6) cw- -C31- -CI4- -031' -134.0 (15)
C2— C1— C14— C13 0.2(6) CI3'- -C31- -CI4- -C3f 87.1 (11)
C18— C1— C14— C13 -170.9(3) CI1- -C31- -CI4- -C3V -79.0(12)
C12— C13— C14— C1 -148.5 (4) cir- -C31- -CI4- -C3V 101.0(12)
C26— C13— C14— C1 93.6 (4) CI3- -C31- -CI4- -C3V -36.5 (11)
C4— C13— C14— C1 -28.1 (5) CI2- -C31- -CI4- -C3V 24.5 (8)
C12— C13— C14— C15 36.8(4) C31 — C31 — CI4- -C¼< 134.0(15)
C26— C13— C14— C15 -81.0(4) CI3'- -C31- -CI4- -C14' -138.9 (10)
C4— C13— C14— C15 157.2(3) CI1- -C31- -CI4- -CI4' 55.0(7)
C1— C14— C15— C16 -11.2(5) cir- -C31- -CI4- -CI41 -125.0(10)
C13— C14— C15— C16 163.4(3) CI3- -C31- -CI4- -CI4' 97.5 (8)
C14— C15— C16— C17 40.8(5) CI2- -C31- -CI4- -CI41 158.5 (9)
C15— C16— C17— C27 168.9(3)
Symmetry codes: (i) -x+1 , -y+2, z. Table 5 gives the Hydrogen-bond geometry (A, °)
Table 5
D— -A D— H H—A D-A D—H-A
01— H1 -04' 0.82 1.81 2.621 (6) 168
03— H3-02i 0.82 1.85 2.670 (4) 176
C22— H22B-01 0.96 2.56 3.380 (5) 144
Symmetry codes: (i) -x+1 , -y+2, z.
Based on the evidential data from IR and 1H NMR spectra of compound suggested the presence of C-3,4-seco-olean-4,12-diene carbon skeleton in the structure and it was characterized as (3,4-seco-olean-4(23),20-diene-3,30-dioic acid which is commonly known as koetjapic acid (see Fig. 1 ). The structural formula of the compound is C3oH4604. The complete structural characteristics were further confirmed by X-ray crystallographic studies.' ii. Anti-proliferative efficacy of koetjapic acid (KA) Cytotoxic potency of koetjapic acid (KA) was evaluated using MTT assay on HUVEC, HCT 1 16, MCF7, MDA-MB-231 and Hep G2 cell lines. KA exhibited strong cytotoxicity against colorectal cancer (HCT 1 16) cell line with IC50 (concentration of test substance to achieve 50% inhibition) 18.88 g/ml compared with other cell lines (P = 0.000). However it showed mild toxicity against other cell lines tested, although it was able to inhibit their proliferation at higher concentrations (see Fig. 4). The inhibitory effect of KA on HCT 1 16 was comparable with the positive controls used. Table 1 depicts the IC50 values of all tested compounds on the indicated cell lines. iii. Colony formation assay
To confirm the cytotoxic effect of KA on HCT 1 16 cells; a colony formation assay was conducted. The effect was found cytotoxic rather than cytostatic as evident by a decrease in a clonogenic survival. The PE was 38.1 ± 4.6%. At 50 and 75 pg/ml SF was almost 0.0%. However at lower concentrations (12.5, 25 pg/ml) the survival rates were 55.3 ± 1.6% and 41.9 ± 1 .4% respectively. The calculated IC50 was 15.78 ± 1.2 g/ml. Figure 5 depicts the survival rates of HCT 1 16 cells upon treatment with KA. iv. Koetjapic acid induces apoptosis by activating caspases 3/7, 8 and 9
An attempt was made to identify whether the KA induced inhibition of cell proliferation was due to induction of apoptosis. After three hours treatment, KA induced the appearance of caspases activities in HCT 1 16 cells in presence of Caspase-Glo reagent. From the results it is conspicuous that, KA activates the initiator caspases, i.e., caspases 8 and 9. The activity of the initiator caspases in treated cells was about 3 fold higher than the control. Furthermore, KA was found to be more specific for the induction of caspase 8 activity, since the induction of caspases 9 was less significant than the caspase 8. Fig. 6 illustrates the induction of caspases activity by koetjapic acid. v. DNA fragmentation Induced by koetjapic acid
To investigate the relationship between the anti-proliferative activity and the koetjapic acid-induced activity of apoptosis, DNA was extracted from the treated HCT 1 16 cells and analyzed by DNA agarose gel electrophoresis. An obvious, dose-dependent, laddering pattern was observed for the concentration range from 10 to 40 g/ml (see Fig. 7). The presence of these oligonucleosomal DNA fragments indicates that the human colorectal (HCT 116) carcinoma cells had undergone apoptotic cell death under the influence of koetjapic acid (KA). However, KA did not cause any DNA fragmentation at a concentration of 10 [ig/m\. vi. Morphological Modifications and nuclear condensation induced by koetjapic acid
The apoptotic morphological events occurring in the nuclei after incubation of HCT 1 16 cells with koetjapic acid (KA) was also studied. No significant morphological changes were observed in nuclei incubated in the absence of KA. At higher concentrations of KA 30 g/ml the nuclei displayed the typical apoptotic changes in the chromatin structure appearing remarkably condensed at the nuclear periphery. Chromatin condensed progressively, in a dose dependent manner (see Fig. 5), to eventually either cluster against the nuclear periphery or to display the characteristic half-moon features observed at higher concentrations. In presence of KA at 20 g/ml concentration the nuclei started to shrink and the chromatin started to collapse into high density structures. The apoptotic index of koetjapic at 40 g/ml with HCT 1 16 cells was 27.3 % and 60.3 % after 6 hours and 9 hours of incubation respectively, while at 20 pg/rnl the apoptotic indexes were 12% and 30% after 6 and 9 hours respectively (see Fig.8). vii. Koetjapic acid reduces mitochondrial potential
To investigate whether the apoptosis induced by koetjapic acid (KA) in HCT 1 16 cells involved the loss of mitochondrial integrity, the mitochondrial membrane potential in HCT 1 16 cells was evaluated by visualizing the uptake of the lipophilic cation dye rhodamine into mitochondria. The loss of mitochondrial membrane potential (ΔΨ) is a hallmark for apoptosis. When the mitochondrial membrane potential decreases, the rhodamine 123 uptake by the cells increases and the florescent signal intensifies exponentially. Results showed an obvious intensification of fluorescence in cells treated with KA (20 Mg/ml) compared with control samples, which suggests a loss in mitochondrial membrane potential. Furthermore, the fluorescent intensity increased with treatment duration of the effect of KA on mitochondrial membrane potential (see Fig. 9). viii. Koetjapic acid modulated the gene expression in HCT 1 16
Gene expression profile has been changed greatly by the koetjapic acid (KA), as shown in Fig. 10. Cancer pathway study revealed that, KA has an extensive influence on up-regulation of NF- B ( 1 .17 fold) . On the other hand the compound caused significant down-regulation of five genes MAPK JNK, MAPK/ERK, HIF, MYC/MAX and Wnt (0.71 , 0.56, 0.59, 0.8, 0.78 fold changes respectively). Remarkable statistical significance was obtained, as shown in Fig. 7. ix. Koetjapic acid inhibits the sprouting of microvessels in rat aorta
In order to assess the anti-angiogenesis ability of koetjapic acid (KA), an ex vivo rat aortic ring assay was conducted. This model was shown to be quite well correlated with in vivo events of neovascularisation. In this assay, the rat aortic endothelium exposed to a three-dimensional matrix containing angiogenic factors switches to a microvascular phenotype generating branching networks of microvessels. As shown in Fig. 1 1A, microvessels grew out extensively from the rat aorta in the control using 1 % ethanol as a vehicle. Whereas, KA significantly inhibited the sprouting of rat aortic microvessels (see Figs. 1 1 B and 1 1 C) with IC50 16.8 pg/ml. The anti-angiogenic effect on explants of rat aorta was significantly dose dependent (P<0.05). There was complete inhibition of vascularisation at the 40 pg/ml which is the IC50 for HUVECs proliferation. At a concentration of 20 g/ml KA , which is the highest non-toxic dose on HUVECs proliferation, the observed inhibition of rat aortic microvessels was around 50 %. Suramine was used as a positive control (see Fig. 1 1 D). x. Inhibitory effect of koetjapic acid on HUVECs migration To evaluate the inhibitory effect of koetjapic acid (KA) on endothelial cell migration process, in vitro wound healing assay was conducted. This assay represents an important step in the formation of new blood vessels, and is a straightforward and economical method to study the cell migration phenomenon. A scratch wound was created on the monolayer of cells. KA (20 Mg/ml) inhibited HUVECs migration drastically by 27.3 % after 12 hours and 23.6 % after 18 hours (P< 0.001 ). Even at lower concentration of KA (10 pg/ml) a significant (P < 0.05) inhibitory effect of 6.9 and 10.6% inhibition after 12 and 18 hours respectively (see Fig. 12). xi. Inhibitory effect of koetjapic acid on tube formation in HUVECs
To characterize the anti-angiogenesis activity of koetjapic acid (KA), it was first determined whether KA inhibited the growth factors induced tube formation of endothelial cells on Matrigel, as shown in Figs. 13A-E that depict a dose- dependent inhibition of tube formation by HUVECs. Endothelial cells formed tube- like networks (see Fig. 1 1 A) within 6 hours, which might, in part, reflect the process of angiogenesis. At a concentration of 40 pg/ml (see Fig. 13C), the KA absolutely abrogated endothelial tube formation by reducing the tube-like structure both in width and in length. Endothelial cells rounded up and rendered network structures incomplete and broken in the presence of KA (see Figs.13B-D). The activity of KA (IC50 = 15.01 pg/ml) was comparable with that of standard drug suramin (Fig. 13D). xii. Koetjapic acid inhibits the production of VEGF signal
The observed inhibition of VEGF-induced endothelial cell tube formation and migration by koetjapic acid (KA) was confirmed by quantifying VEGF 165 levels in endothelial cell lysates. An ELISA-based kit (RayBio, USA) was used to quantitatively measure the production of VEGF protein signal, a measure of angiogenesis in endothelial cells. KA caused significant inhibition of VEGF production from endothelial cells. ELISA measurements indicated that control cells showed higher levels of VEGF 165 (378 pg/ml) in cell lysates than cells treated with 20 pg/ml KA (362pg/ml) (P<0.001). xiii. In- vivo CAM assay Vascularization in chick embryo was significantly inhibited by the 100 pg koetjapic acid. Images of two chorioallantoic membranes are shown in Figs. 14A and 14B. The vasculature pattern formed by the blood vessels in CAM of control group treated with vehicle was normal. The primary, secondary and tertiary vessel with the dendritic branching pattern, which is characteristic of CAMs, was well established in control CAMs and can be seen clearly in Fig. 14A. On the other hand, the 100 pg KA treated CAM (1 mg/disc) showed the distorted architecture in vasculature, as shown in Fig. 14B.
Discussion
Chemotherapeutic agents act primarily by inducing cancer cell death via apoptosis. Nevertheless, there are many cancers that are intrinsically resistant to apoptosis, but susceptible to angiogenesis inhibition. This state of affair highlights the importance of angiogenesis inhibitors as part of the classical chemotherapy regimen. In the present invention, the inventors found that koetjapic acid, may be a promising new anticancer agent for human colorectal cancer by acting both as apoptotic inducer and angiogenesis inhibitor.
Koetjapic acid, a bioactive secotriterpene, has been described as a β-polimerase inhibitor. Antitumor agents, particularly the DNA polymerase inhibitors, act by inhibiting cell growth and/or causing apoptotic cell death in various cancerous cell lines.
The present invention shows that koetjapic acid (KA) has significant anti-cancer activity towards HCT 1 16 colorectal carcinoma cells via the apoptotic pathway. Apoptosis, can be triggered by a variety of stimuli, including cell surface receptors, signaling molecules, enzymes, gene regulating proteins and mitochondrial response to chemical stress. Among them are the caspase-cascade signalling system, regulated by various inducers of apoptosis. Caspases are a class of cysteine proteases that includes several key player. The caspase 8 and 9 are the gate keepers of the caspase apoptosis cascade. Caspase 8 forms as the initial caspase that is involved during a response towards signals by receptors with a death domain. Caspase 9 is triggered via mitochondrial damage. The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then leads to the activation of caspase 9. Caspase 3 and 7 are downstream caspases that are activated by the upstream proteases and act individually to cleave cellular targets. In the present invention, HCT 1 16 cells treated with KA displayed elevated level of caspases activity. Here, the inventors found that both caspases 8 and 9 were induced by KA. These findings are complimentary with work done by other researchers who have shown that caspase 8 alone is insufficient to execute cell death so it concomitantly interacts with the intrinsic apoptotic pathway by causing damage to mitochondrial membrane which in turn releases cytochrome-c. Caspase 3-like activity has been detected in apoptotic cell death induced by a number of chemotherapeutic drugs. It has also been reported that caspase 3 and 7 are associated with DNA fragmentation and apoptotic cell morphological changes. Indeed, many chemotherapeutic agents induce apoptotic cell death by causing DNA fragmentation.
The present inventors find that koetjapic acid (KA) caused a dose dependent DNA fragmentation on HCT 1 16 cells. The compound also caused nuclear morphological changes and chromatin condensation in HCT 1 16 cells leading to apoptotic bodies formation.
Failure in formation of formazone crystals in MTT assay and elevated levels of caspase-9 and -3 in HCT 1 16 cells, suggest koetjapic acid (KA) induced mitochondrial damage. Significant increase in rhodamine fluorescent intensity after treatment with KA provided an additional evidence of loss of mitochondrial membrane potential which is required for cytochrome c release and activation of the intrinsic pathway. This clearly implies that, KA can induce mitochondrial permeability transition through an increase in intracellular cation concentration, and can cause a significant loss in mitochondrial membrane potential which is an important prerequisite in the activation of apoptosis.
Discovery of the oncogenes, paved the way for the emergence of a new generation of cancer therapies, those targeted at specific signalling molecules. The signalling pathways controlling cell growth and differentiation are almost invariably altered in cancer. These interconnected pathways are being deciphered, but understanding the alterations that lead to cancer and correcting them is a substantial challenge.
To detect the potential genes that contribute to apoptosis in HCT 116 cells caused by koetjapic acid (KA) induced cytotoxicity, the present inventors assessed the early changes in genetic prolife of a series of genes controlling cell cycle and apoptosis such as Wnt, NF-κΒ , HIF, MYC/MAX and MAPK pathways.
Wnt signalling is important in maintaining sternness in normal colon stem cells and hyper-activation of this pathway is common in most colon cancers. Using Wnt signaling activity as readout, the inventors found that koetjapic acid (KA) caused down-regulation of Wnt pathway through the by affecting the formation of β- catenin/Tcf4 complexes. Many studies have highlighted the important role Wnt-β- catenin pathway in colorectal cancer and their potential as cancer drug target.
Colorectal cancers are highly angiogenic and were the first tumor cell type to exhibit significant responses to anti-angiogenic agents both in vitro and in vivo. Including the drugs for colon cancer, a growing number of anticancer agents have been shown to inhibit hypoxia inducible factor (HIF) gene activity. For many of these, the mechanism of action has been established and involves a reduction in HIF-1 mRNA or protein levels by suppressing the HIF gene expression. Hypoxia is the principal trigger to stimulate VEGF gene transcription and subsequently to angiogenesis. The present invention shows that koetjapic acid (KA) caused significant reduction of HIF gene of treated colon cancer cells. The results also show that KA inhibited microvessel formation in rat aorta, and thwart endothelial cell proliferation and migration which all essential for angiogenesis. It is noteworthy to mention that Wnt signaling is also involved in the regulation of endothelial cell proliferation and migration during angiogenesis, and HIF overexpression is associated with pathologic angiogenesis. It is likely that the observed ex vivo and in vitro anti-angiogenic potencies observed in the present invention are the consequence of koetjapic acid (KA) induced Wnt and HIF down-regulation. The antiangiogenic activity of koetjapic acid (KA) is further supported by the in vivo studies employing fertilized chicken embryo (CAM) where compound caused marked inhibition in blood vessel formation.
Based on numerous basic and clinical observations, NF-κΒ suppression is often interpreted as a potential future of chemotherapeutics to curb tumourigenesis. Nevertheless, recently this proposal has been challenged by several tumour models, particularly in colorectal cancer, in which NF-κΒ activation has been hypothesized as a safeguard against tumourigenesis. In addition to regulating cellular responses to cytokines and pathogens, the NF-κΒ pathway plays an essential role in controlling cellular growth properties and apoptotic cell death. In the present work, findings showed a significant upregulation in NF-κΒ gene in koetjapic acid (KA) treated HCT 1 16 cells. This result correlates with that of aspirin on colon cancer cells. Aspirin, a non-steroidal anti-inflammatory drug, induces apoptosis in colon cancer cells by upregulating the NF-κΒ signalling pathway, koetjapic acid (KA) is reported to have an anti-inflammatory property, and most probably, similar to the aspirin it may stimulate NF-κΒ through nuclear translocation of NF-κΒ complexes.
Another important protein in the cancer disease in the Myc cellular protein. Myc has a vital role in the proliferation of cell, and is crucial for the maintenance of stem cell compartments and the balance between self-renewal and differentiation in multiple tissues, including the intestinal crypts. Myc is deregulated and overexpressed in most cancer cells, where it impair the diverse intracellular and extracellular regulators of normal cell proliferation and render it to cancerous. Aberrant Myc expression in most human cancers is usually not due to mutation in the Myc gene itself but a consequence of its induction by 'upstream' oncogenic signals, thus, inhibition of Myc gene evolved as a candidate in chemotherapy. In the present study, results show that koetjapic acid (KA) significantly suppressed Myc pathway expression. This provides an additional complementary and substantial evidence of the compound's anticancer efficiency.
In addition to the pathways discussed above, c-Jun NH(2)-terminal protein kinases (JNK) and extracellular signal-regulated kinases (ERK) pathways were also investigated, which are the active members of mitogen-activated protein kinases (MAPK) family. MAP kinases are Serine-threonine protein kinases involved in cellular responses to mitogen stimulation, environmental stress, pro-inflammatory cytokines, and apoptotic stimuli. Apart from these, recent studies have shown that MAPK cascade plays a central role in angiogenesis by specifically inducing VEGF mRNA expression. Similarly, another study reported that MAP kinase signaling pathway and HIF pathway cooperatively take part in the control of angiogenesis, notably in the expression of VEGF signal. Several studies have shown that down- regulation of the expression of MAPK gene products, leads to the down-regulation of HIF signalling pathway that in turn ultimately leads to induction of apoptosis, cell cycle arrest and suppressing the transcription of VEGF. Because the MAPK cascade controls growth and survival directly at the level of tumoral cells and secondarily by its paracrine action via VEGF secretion, it represents a target of choice for therapeutic intervention in cancer. Koetjapic acid (KA) caused a drastic down-regulation of these MAPK genes in treated cells that further supports its anti- angiogenic and anti-apoptotic effects by suppressing the pro-proliferation genes. Conclusion
The result of this study highlights the potential role of koetjapic acid (KA) as a potential anticancer agent, koetjapic acid (KA) causes apoptotic cell death of HCT 1 16 colon cancer cells by inducing the activation of caspases. The alterations of mitochondrial membrane potential may lead to cytochrome C release and initiate apoptosis cascade in targeted cells. In line with, it was found that koetjapic acid (KA) exhibits anti-angiogenesis property by inhibiting ex vivo sprouting of rat aorta microvessels and in vitro proliferation, migration, tube formation and VEGF production in endothelial cells. Koetjapic acid (KA) also inhibited angiogenesis in vivo in fertilised chicken egg embryo. The involvement of Wnt, HIF, MAPK/ERK/JNK, Myc/Max and NF-kB signalling pathways in koetjapic acid (KA) induced apoptosis concludes that koetjapic acid (KA) induces apoptosis in human colorectal cancer (HCT 1 16) cells either by down regulating pro-proliferation genes or by upregulating the tumor suppressor genes. Hence, koetjapic acid (KA) may be useful in variety of angiogenesis related ailments as well as large number of other form of neoplasm.

Claims

1. A composition consisting essentially of an amount of a compound effective for inhibiting colorectal cancer cell growth, the compound having the following structure:
Figure imgf000067_0001
a chemical named seco-A-ring oleanene triterpene.
2. A composition according to claim 1 further comprising a pharmaceutically acceptable carrier.
3. A composition according to claim 1 , wherein the compound is isolated and purified from Sandoricum koetjape plant extract.
4. A composition for inhibiting cancer cell growth, consisting essentially following structure:
Figure imgf000068_0001
also named seco-A-ring oleanene triterpene, wherein the cancer is colorectal cancer.
5. A composition according to claim 4 further comprising a pharmaceutically acceptable carrier.
6. A composition of claim 4, wherein the composition is obtained by a method comprising the steps of: i. extracting plant materials of Sandoricum koetjape or plant powder with one or more organic solvents to obtain an organic extract; ii. collecting the organic extract;
iii. drying and the organic extract to form a Sandoricum koetjape extract powder;
iv. dissolving the Sandoricum koetjape extract powder in one or more organic solvents to form a precipitate;
v. collecting the precipitate; and
vi. washing and purifying the precipitate to obtain crystallised seco-A- ring oleanene triterpene or koetjapic acid.
7. A composition according to claim 6, wherein the plant materials include leaves, branches, stems, fruit-stems, roots and barks.
8. A composition according to claim 6, wherein the organic solvent includes hexane, methanol and acetone.
9. Use of a composition consisting essentially of a compound having the following structure in the manufacture of a medicament for inhibiting colorectal cancer cell rowth.
Figure imgf000069_0001
10. A use according to claim 9, wherein the compound is isolated and purified from Sandoricum koetjape plant extract.
PCT/MY2012/000009 2011-08-22 2012-01-30 Composition comprising sandoricum koetjape extracts and uses thereof WO2013028051A1 (en)

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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
HU, H.-Y. ET AL.: "Identification of Small Molecule Synthetic Inhibitors of DNA Polymerase beta by NMR Chemical Shift Mapping", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, 2004, pages 39736 - 39744 *
ISMAIL,1. S. ET AL.: "Ichthyotoxic and Anticarcinogenic Effects of Triterpenoids from Sandoricum koetjape Bark", BIOLOGICAL AND PHARMACEUTICAL BULLETIN, vol. 26, 2003, pages 1351 - 1353 *
KANEDA, N. ET AL.: "Plant Anticancer Agents, L. Cytotoxic Triterpenes from Sandoricum Koetjape Stems", JOURNAL OF NATURAL PRODUCTS, vol. 55, 1992, pages 654 - 659, XP008004656, DOI: doi:10.1021/np50083a016 *
NASSAR, Z. D. ET AL.: "Antiangiogenic properties of Koetjapic acid, a natural triterpene isolated from Sandoricum koetjaoe Merr", CANCER CELL INTERNATIONAL 2011, vol. 11, 27 April 2011 (2011-04-27), pages 6 OF 8 *
NASSAR, Z. D. ET AL.: "Koetjapic acid, a natural triterpenoid, induces apoptosis in colon cancer cells", ONCOLOGY REPORTS, vol. 27, 30 November 2011 (2011-11-30), pages 727 - 733 *
RASADAH, M. A. ET AL.: "Anti-inflammatory agents from Sandoricum koetjape Merr", PHYTOMEDICINE, vol. 11, 2004, pages 261 - 263, XP004957037, DOI: doi:10.1078/0944-7113-00339 *
SUN, D.-A. ET AL.: "`DNA Polymerase beta Inhibitors from Sandoricum koetjape", JOURNAL OF NATURAL PRODUCTS, vol. 62, 1999, pages 1110 - 1113 *
TANAKA, T. ET AL.: "`New Multiflorane-Type Triterpenoid Acids from Sandoricum indicum", JOURNAL OF NATURAL PRODUCTS, vol. 64, 2001, pages 1243 - 1245 *

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