WO2001049306A1 - Balanites aegyptiaca extracts for treatment of hiv/aids and leukemia - Google Patents

Abstract

This patent application deals with the revealing of the effectiveness of Balanites aegyptica bark aqueous extract in treatment of both Aids and Leukemia. It also reveals the magnitude of safety of the aqueous extract. An oral administration of the aqueous extract (30 % w/v given at 100ml every 8hrs for 30 days) for the treatment of Hiv/Aids patients have shown excellent results. The same was given to patients with Leukemia and a good increase in platelets (25000 weekly), (TwBc 500 weakly) and a normal blood differential reading after one month. The treatment was very well tolerated by the patients without side effects or complication.

Description

BALANITES AEGYPTIACA EXTRACTS FOR TREATMENT OF HIV/AIDS AND LEUKEMIA

General Introduction :

Dalanites aegyptiaca (L.) Del is one of the very useful trees in the Sudan. Various parts of the tree arc reputed locally lo exhibit economical importance and medicinal fol loric uses . The species & family :

Dalanites aegyptiaca (L.) Dcl., is the only species of the genus Dalanites present in the Sudan (Andrews 1952). The genus Dalanites was placed in different families by various taxonomists, but recently it is recognized by Takhtajan (1969), due to its peculiarity, as the only genus in the family Balauitaccac. According to him, the classification of the family is as follows :

Division : Magnoliophyta

Class : agnoliatae or Dicotyledons

Subclass : osidac

Superorder : Rutanae

Order : Gcranialcs

Family : Balanitaccae

Geographical Distribution :

D. aegyptiaca is a large savanna tree widely distributed throughout Afrcia, a long the tropical belt from Tanzania in the East to Ivory Coast in the West. It is also found in the relatively drier regions of Northern Africa from Mυritania to Nigeria and Ghana, to Egypt, across Palestine, Saudi Arabia and India. The drier regions of Kenya, Uganda and Zaire, carry scattered open forests of D .aegyptiaca (Suliman & Jackson (1959) . In the Sudan the tree is widespread throughout the Northern and Central Provinces (Wickcns 1976) . Habita :

The tree is common in the low Rainfall Savanna and semi-desert vegctational types (Harrison & Jackson 1958) . It grows in various soil types such as clay, dark cracking caly, sand, hard-surfaced sandy clay, etc.. It flourishes in habitats of clay soils receiving 500- 1000 mm of annual rainfall as well as on sandy clay soil where rainfall exceeds 250 mm. annually. However, the most luxurious Dalanites forests are typically found on slightly elevated dark cracking clay under a rainfall of 500 mm and upwards annually .

Charactcrstics of the genus Dalanites Diagnostic Characters of the genus :

Habit woody; leaves alternate, 2-foliate; spines green; flowers with free sepals and petals, stamens twice as many as the petals, disc lobed, pistil with 5 locules and a single style (Burger 1967) . Common species of the genus :

- D.aegyptiaca (I.) Del.

- D.aegyptiaca (L.) Del., var. pallida Sondi.

- D.aegyptiaca Del., var. quarrel (Dc Wild) G.Gilbcrt .

- D .pedicellaris Mildb. & Schlccht., subsp. somalcnsis (Mildb. & scheicht.) Sonds .

- D.rotundifolia (van Tieg .) Blatter .

- Λvilsotuana Dawc et sprangue . Synonyms of the genus :

- A gialid Adans.

- A gihalid Juss .

- A gialida O.Ktze .

- A giella van Tiegh . Chracterstics of the species Dlatiiies aegyptiaca (L.) Delile. In Dcscr. Egypt, Hist, Nat. 2:221, to. 28, 1(1913). Synonyms :

- Ximenia aegyptiaca L.

- A gialida senegalensis van Tiegh .

- AMarteria van Tiegh .

- A.tombo ctensis van Tiegh .

- Dalanites zizyphoides Mildbrs. & schlechter .

- D.roxburghil Planch .

- Jerox G. Don.

- D.aegyptiaca Wall . Vernacular Names :

Arabic : Heglig (Tree), Laloub (fruits)

English : Thorn tree, Soapbeπy, Desert date, Egyptian balsam, Indian Dalanites,

Mexican Dalanites, Zachuni oil, Kernel oil, Microbaians (fruits) . French : Datticr du desert, Hagueleg, Dalanit d' Egypt. Egyptian : Shashul Sudanese : (local languages) . (Fur) : daay or daaynga, pi. keingu (Hadcndowa) : Sassud, Shashot, Shashoba (Nuba) : (J.Daicr): Korak

(DiUing & J. Gliulfan) : Tira (J.Elliri) : Kuri (kadugli) : N^! Dumusso (Bari) : Lallok and Lumili (Golo) : Sorongo (Hamcg) : K a, Ga (Burn) : Toan (Jur, Nuer,

Kaka, shilluk) : Tu, Tau

(Nuer) : Fiath

(Beni Amer) : Qog, G' ogat

(Madi, Lugbari) : Loba, Logba

(Λcholi) : To

Habit :

Small spincscnt evergreen savanna trees, up to 15 in high (rarely shrubs); girth 175 cm at 50 cm above the ground (Fadl 1982), crown flat, rounded or irregular, diameter up to 8 m; with fluted bole. Taxonoinic dcsriptiou : a. Bark : rough, grey to dark brown; scales long, thick, prominent, ragged; fissures long, deep, vertical; slash pale yellow . . b. Branches & Branchlεts : green or greyish, stiff and briule, drooping . c. Spines : simple, straight and forward-directed, stout, rigid, green, alternate, supra-axillary, up to 8 cm long . d. Leaves : palmately compound, two-foliate, alternate, 2.8 -5.0 em long, stipules absent . e. Leaflets .' 2, subsessile; lamina variable : broadly elliptic, spaUiulatc, obovate to 'τbiπιln.r- rhomboid/, 2,5 -5-P *{• 2-2- cm.

orbicular- rιιυιuυυ-u, _^.._{^ ^}._^ --. f. Inflorescence : supra- xillary clusters or rarely subraccmosc . g. Flowers : bisexual, regular, with spicy scent, 1.0 -1.5 cm across . h. Sepals : 5, free, imbricate, 4-6 x 3.5- 4.0 mm., densely pubescent outside, with IOΠR silky whitish hairs inside . 1. Qvary : superior, 5-locular, densely covered with silky hairs; placcntation axilc. m. Ovules : 1 in each loculous n. Fruits : drupe (stone-fruit); oval, pear-shaped, up to 5.0 x 2.5 cm. green turning yellow or brown; composed of the following layers : a. epicarp : outer part, leathery, smooth or wrinkled . b. mesocarp : middle part, yellow-brown sticky flesh, oily, gummy with bittcr- , sweet taste . c. cndocarp : inner part, hard-pointed, woody and surrounds the kernel . d. Seeds : ovoid, testa sub-fibrous, non-cndospeπnic .

Regeneration :

D. aegyptiaca has a high regeneration power. Regeneration is usually by seeds (Suliman & Jackson 1959) though it is a good coppiccr as its cut ste s profusely coppice. Its roots when exposed or injured produce aerial shoots developing into daughter plants (Amalraj 1987). The tree regenerates readily after lopping or heavily browsing, although finally acquiring adwarf bush growth form (von Maydell 1986) . Propagation :

D. aegyptiaca propagates mainly by direct seeding, but also by root suckers and cuttings. A mature tree may yield about 10,000 fruits per year. First fruit yields may be expected after 5 to 8 years. The tree can attain an age of more than 100 years (von Maydell 1986) . Leafin :

Evergreen tree, but can be deciduous or partially deciduous in low rainfall areas or in the dry seasons. Flowering season :

November and sometimes in April . Fruiting season :

December and January, occasionally later from March to July, and exceptionally in August in the Red Sea Area .

Pollination :

The species is both self-ami cross-compatible. Cross pollination is mainly by flies and bees . dispersal of fruits :

The fruits which are indehisccnt and edible, specially the sticky mesocarp, are widely dispersed by animals, human beings & birds. The endocarps arc indigestible and thus will pass the intestinal tracts of the animals without losing their viability ,

Chemical constituents :

- Fruit : The fruits of D.aegyptiaca were found to contain isorhamnetin 3-rutinoside and 3-rhamnogalactosidc (Maksoud and Al-Hadidi, 1988) . The alcoholic extract of the pulp and kernel contained sterols, terpenεs and saponins as predominant compounds where as tannins, alkaloids and resins were found in slightly small amounts (Abdcl Rahim et al, 1986). Five saponins were isolated from the pulp and named as Balaniti- sins A,B,C,D and E. (Varshney et al, 1 77, Varshney and Jain, 1979). Two other Saponins named as Balanitisins F and G were isolated from the kernel (Varshney and Vyas, 1982). The total saponin content was found to be 7.2% in the mesocarp and 6.7% in the kernel (Watt and Breyer-Brandwijk, 1962). The oil extracted from the kernel constituted 44-51% w/w and is composed mainly of triglycerides and with small quantities of diglyccridcs, phytosterols, sterolcstcrs and tocophcrυls. The oil contains palmitic acid 10-12% stearic acid 9-10%, oleic acid 30-40% and linoleic acid 40-48% w/w (Abu-Al-Futuh, 1983) .

Leaf : six flavonoid glycosidcs identified as qucrcclin 3-glucosidc, quercctin 3- rutinosidc, 3,7, digluscoside and 3-rhamnogalactoside of isorhamnetin, were isolated from the leaves and branches of the plant (Maksoud and El-Iladidi, 1 88) . - Stem bark : Three saponins (Yamogcnin muclcus) known as Balanitisins 1 , 2 and 3 were isolated from the East African specimen of D.aegyptiaca (Liu and Nakanishi, 1982). From the Indian specimen was isolated an antifeedant sponin made up of dios- gcnin/ yamogcnin and the sugars glucose and rhamnosc in the ratio of 3: 1 (Jain, 1987). In addition to the above saponins two other saponins were isolated and identified as the previously known saponins deltonin and protodcltonin. A Furanocoumarin, bergapten and a dihydrofuranocoumarin, marmesin were isolated from the chloroform extract of the bark (Ahmed et al, 19 1). A new scsquitcrpcne named as balanitol was isolated from the bark of die Indian specimen (Cordano et al, 1 78) .

- Stem wood: Balanitisin 1 was isojalcd rom the stem wood of the Indian specimen (Varshney and Vyas, 1982) .

- Root: Dahuiitisins 1 ,2 and 3 were isolated from the East African specimen (Liu and nakanishi, 1982) and the presence of alkaloids was reported in the root bark (El- Khicr, 1987) .

- Root wood: Balanitisin I I was isolated from die root wood of the plant (Varshney and

Vyas, 1982) . Medicinal Folk-uses :

- Whole plant : anthelmintic, purgative, boils, leucυderma, herpes, vermifuge, malaria, emetic, wounds, syphilis, colds, liver and spleen problems, aches and febrifuge.

- Bark : stomachaches, mental diseases, epilepsy, yellow fever, syphilis, jaundice, fumi- gant to hpal circumcision wounds.

- Shoot : chewed into a paste for dressing wounds .

- Leaves : cleans malignant wounds .

- Fruit and kernel : mild laxative, antidote to arrow poison, purgative, vermifuge, stomach ailment, bilharzia .

- Kernel oils skin disease, rheumatism .

- Root : malaria . Economic uses :

- bark : yields a strong fibre and soap for washing clothes .

- twigs : for toothbrushes when frayed .

- leaves : boiled to remove the bitter taste and then eaten (in times of famine) .

- Fruits and roots : powdered and used as a soap .

- fruit kernel : yields an oil which is used as an unguent .

- wood : for writting slates, bowls and other utensils, mortar, pestles, implement- handles, joinary, agricultural implements, local furnitures, window sills, walking- sticks and bent wood chaus. It provides excellent firewood and charcoal, with a calorific value of 4600 K cal./kg .

Some Pharmacological and Toxicological Studies on Dalanites aegyptiaca (L.) Del. Bark

1. Introduction

D.aegyptiaca (F. Dalanitaceae), a tree known as llcglig (Arabic), Thorn tree or Desert date (English) is widely distributed along the tropical belt of Africa, in Sudan, Chad, Nigeria, Tanzania, Gambia, Upper Volta, North Guinea, Kenya & Uganda (Suliman and Jackson, 1959). The Indian tree D.roxb rg ii is regarded as identical to D. aegyptiaca (Hard an, 1969). The tree has many folk uses in various African countries. The fruit is used as fumigatory in liver diseases in Chad (Croach, 1962; Watt and Brcycrs-Brandwijk, 1962), and as a purgative ami sucked by schools children as a confectionery in Sudan (Λbu-El-Futuh, 1983). In Tanzania the bark is used in treatment of syphillis, round worm infections and as a fish poison (Bailey, 1962). The root, bark, seed kernel, fruit and branch were lethal to snails, iracidia and ccreariac of schisto- somes (Archibald, 1933; Watt and Breyers-Drandwijk, 1962; Bashir et al, 1984). The aqueous extract and saponins isolated from kernel cakes have a potent larvicidal activity (Sarroug et al., 1988) and anti-bacterial activity (Bashir et al., 1 84). In the Sudanese folk medicine the aqueous extract of the bark is widely used as anti-jaundice. Thus it is thought of interest to subject the plant bark for intensive pharmacological, toxicological and clinical studies .

2. Material and Methods A. Aqueous extract

The bark of D.aegyptiaca was removed, air-dried in shade, coarsely powdered and kept in air-tied containers. The powder (15,30,60 g) was added to 100 ml distilled water in a 500 ml-bcakcr. The content was boiled for 20 minutes (min), and allowed to cool, filtered through a cotton wool and rclilteicd using a Whatman filter paper. The filtcratc was adjusted to 100 ml by adding distilled water_* This filterate was freshly prepared everyday before experimentation . In another series of experiments, the filtcrates were freeze- ricd and the dried substance was kept in dcsicators. The aqueous solution of this dried substance was prepared immediately before experiments .

B. Ethanolic extract

Two hundred g of the coarsely powdered bark were extracted by ethanol (95%) in a soxhlct after defatting with petroleum ether (6O-8O0C). The ethanolic extract was cva- portcd to dryncss under vaccu and kept in a desicator. On the day of experimentation the extract was rcsuspcndcd in normal saline and filtered. The filtcrate was used immediately.

C. Isolated tissues

A number of isolated tissues (rabbit intestine, rabbit aortic strip, rat uterus, rat stomach strip, rat phrenic-nerve diaphragm preparation and perfused rubbit heart) were prepared as described by Kithchcn (1984). The tissues were suspended in their corresponding physiological solutions ic, Tyiodc's, Kreb's, Dcjalυn's or Ringer-Lock's solutions . The isotonic contractions of the above tissues were recorded using T3 isotonic transducer (Bioscicnce, Sheerncss, England) connected to MD2 Washington recorder (Bioscicnce) or Harvard istouic transducer (Harvard,, Kent, Englant) coupled to Harvard Universal Oscillograph (Harvard). The isometric contractions were registered using UFI isometric transducer (Bioscicnce) connected to MD₂ Washington recorder .

The 15% (w/v) aqueous extract was added (1 -2 ml) tυ the above suspended tissues. For higher concentrations of the plant aqueous extract, the freeze dried substance was prepared as described before and added tυ the isolated tissues .

D. Induction of experimental jaundice in rate

Male Wistar rats (250 g) were anaesthetized with clicthylethcr, and an incision was made on the upper right quadrant of the abdomen at the level of the duodenum. The duodenum and part of the intestine were exteriorised and die site of opening of the bile duct into the duodenum was identified. A surgical suture was tied firmly around the bile duct and sectioned before its entery in the duodenum. The organs were replaced back into the abdominal cavity and cuts were sutured. Oxytcuacyclinc ointment was applied to prophylact against infection. Animals were allowed to recover from anaesthesia. Thirty min later, the animals were divided into 4 groups (N =6). Groups 1,2 and 3 were injected (i.p) daily for 3 days with 8 ml/kg B.W. of the aqueous bark extract of strength 15,30 and 60% (w/v), respectively. Group 4 served as a control and injected similarly wiUi distilled water (i.p) . On the fourth day blood samples (5 ml) were taken by cardiac puncture from each rat . The blood was allowed to clot and the scrum was aspirated for determination of bilirubin using diazo reaction as described by Malloy and Evelyn (1937). The azobilirubin produced, was measured spectrophotometrically at 540 nm. The scrum bilirubin content was read directly-after necessary dilution with distilled water-against standard bilirubin curve (0.1-1.6 mg l⁽)0 ml) . E. Toxicity studies (i) Acute toxicity

Wistar rats (150-180 g) of cither sex were used. They were divided randomly into 10 groups (N=6). Group i and 2 saved as control and dosed with distilled water 10 ml/ kg B.W. orally and 2 mi/kg B.W. intraperitoneally, respectively. Groups 3,4,5 and 6 were fed orally with 65, 500, 1000 and 200ϋmg of frccze-dried substance/kg B.W. respectively. Groups 7.8.9 and 10 were injected with 65,500,1000 and 2000 mg of the frcczc-dricd substance/kg B.W., respectively. Oral doses were administered in a volume of 10 ml/kg and i.p doses in volume of 2 ml/kg .. All groups were dosed for 3 conscqua- tive days and carefully watched. Twenty four hours later the rats were killed and die vital organs (heart, lung, kidney, spleen, intestine, liver) were grossly observed . (ii) Lethal dose 50 (LD5₀) in mice

Male and female albino mice (25 g) were divided into 16 groups (N=10). Groups 1-9 were injected (i.p) with various volumes (0.05 -3.2 ml/animal) of various strcngdi of die bark aqueous extracts (15,30 and 60% w/v). Groups 10 -16 saved as a control and injectd (i.p) with distilled water (0.05 -3.2 ml/animal) . In anodicr set of experiments the LD₅₀ was determined using die oral route for die adnunisuation of the bark aqueous extracts . Albino mice (25 g) of both sexes were fasted overnight and divided randomly into 10 groups (N=10). groups 1 -3 were fed widi 0.25, 0.5 and 1.0 ml of die 15% (w/v) aqueous plant extract respectively. Group 4 was fed with 1.0 ml of the 30% (w/v) aqueous extract, while groups 5 and 6 were fed with 1.0 and 2.0 ml of die 60% (w/v) aqueous extract, respectively. Groups 7 - 10 saved as a control and fed with distilled water 0.25, 0.5, 1.0 and 2.0 ml/animal, respectively .

All groups were watched carefully for 24 hours and the number and the percentages of death in each group were calculated . The percentages were converted to probits and the LD50 calculated . (iii) Subclironic toxicity (a): In rats

Wistar rats (150 - 180 g) of either sex were divided randomly into 4 groups (N=10). Group 1 saved as a control and fed orally with distilled water (10 ml/kg B.W.). Groups 2,3 and 4 were fed orally widi 65,325 and 1625 mg of the freeze-dried substance of the aqueous bark extract/kg B.W. respectively. Dosing of annuals in ail groups was continued daily for 3 weeks. A blood sample (2.0 ml) was taken from the orbital sinus at day 0 (before dosing), day 8, day 15 and day 22 of experimentation. The blood i sample was immediately divided into two portions. One portion was used for haeniato- logical examinations i.e. haemoglobin estimation (lib), packed cell volume (FCV), white blood cell count (WBC) and red blood cell count (RBC). The second portion of blood was centrifugcd to separate plasma. The total protein content (g 100 ml), albumin (g/100 ml), urea (mg/100 ml), glutamate-oxaloacetate transaminase (GOT U/L) and die glutamatc-pyruvatc transaminase (GPT u/L) were determined clourimetcrically (Thomas and Chamberlin, 1974; Bio Medrieux, 1979) .

All rats in groups were watched carefully for 3 weeks and killed on day 22 of experimentations for gross observation of the vital organs. (b) Subsc ronic toxicity in Chicks

Seven-day old Bovan Chicks were randomly divided into 6 groups (N=12). Group 1 fed with the normal Chick diet (Control group). Groups 2 and 3 were fed with 2 and 10 (w/v) mixture of the powdered bark with the normal Chick diet, respectively. Group 4 fed orally with the ethanolic bark extract (500 mg/kg) prepared as described before. Groups 5 and 6 were injected with the ethanolic extract 50 mg/kg B.W. (i.p) and 10 mg kg B.W. (i.m), respectively .

Dosing of ail Chicks was continued daily for 4 weeks (toxicity period) . Birds were allowed to recover for 3 weeks (recovery period). Chicks were weekly weighed and lots of 4 birds in each group were slaughtered at week 2, 4 and 7 and of treatment. Blood samples were collected for haematology and serology while the vital organs were grossly examined. The blood cellular elements, and scrum constituents were determined as mentioned before ;

Results arc expressed as mean ± S.E.M. and "t" test or analysis of variance was used where appropriate. The difference was considered significant at P = 0.05.

3. Results :

3.1 Percentage of yield of aqueous extract

The percentage of yield of the D.aegyptiaca bark aqueous cxuaci was found to be 4.09 ±0.1 (N=12). The pll of this extract was 5.50 .

3.2 Isolated tissues

The addition of the 15% (w/v) aqueous exuact of D.aegyptiaca bark (1-2 ml/25 ml -gut badr, N=4) was widiout any effect on rabbit intestine, rabbit aortic strips, rat stomach strip, rat uterus or rat phrenic-nerve diaphragm preparation . S nilary the frcczc- dried sample of die extract (0.2 -10 mg/ml; N=4) did not affect these isolated tissues. Like the 15% (w/v) aqueous extract, the frcezc-dricd substance (0.2 -10 mg bolus injection; N=4) did not change the activity of isolated perfused rabbit heart, however, large doses (25 mg, N=4) inhibited significantly (P< 0.05) the conϋactility and the heart rate but decreased insignificantly the flow rate (table 1 ) . 3.3 Experimental jaundice

The scrum bilirubin level of biliary duct-ligatcd control rat was found to be 11.27 ± 0.25 mg/100 ml scrum, when tested on the fourdi day following ligation (N=6). Treatment of animals with balanitcs extracts 15,30 and 60% (w/v) at the dose level of 8 ml/ kg/day for 3 conscquative days starting on day of ligation, induced dose-dependant significant decreases on the serum bilirubin level. Table 2 depicts the different values of scrum bilirubin and the percentage decreases obtained following the different treatments.

3.4 Toxicity studies 3.4.1 Acute toxicity

Administration of the frcezc-dried substance prepared fro t die D.aegyptiaca bark aqueous extract (65 and 500 mg.kg) by oral or intcrapei itioneal route for 3 conscquative days, was without any noticeable effect on rat behaviour, motility or respirations. All rats were healthy and poslmortum examination showed normal vital organs. Similar results wer obtained when animals were fed orally with 1000 and 2000 g of the frceze- dricd substance/kg. However the injection of large doses (1000 and 2000 mg of the frccze-dricd substance/kg i.p) caused nasal bleeding and death of the rats. All rats in the control groups were normal and healthy .

Administration of the bark aqueous extract to mice in doses up to 19.2 g bark/kg (= 768 mg of the frcezc-dried substance/kg (i.p) did not induce any observable toxicity or death in the animals during the 72 hours following the administration . However injection of doses > 19.2 g bark/kg (i.p) induced deadi during the next 24 hours depending on the dose. The calculated LD₅0 value was 33 g bark/kg i.p ( = 1320 g of the frccze- dried substance/kg) .

Similarly administration of the bark aqueous extract to mice in single doses up to 48 g bark/kg orally (= 1920 mg of the frccze-dricd substance/kg) did not produce any toxicity or deadi in the animals during 72 hours following administration . However when the 60% (w/v) aqueous extract was fed in doses of 0.8 ml and 1.6 ml/mouse at hourly intervals the animals started to die during the 4 -6 hours following start of administration . The LD5₀ was found to be 136 g bark/kg orally ( = 5440 mg of the frcezc- dried substance/kg) . In all these acute toxicity experiments none of the animals that were administered water alone (control groups) died.

3.4.2 Subchronic toxicity in rats

Oral dosing of rats for 21 conscquative days with various concentrations of the frcezc-dried substance prepared from the bark aqueous extract (65,325 and 1625 mg kg), was without any observable toxic effect on the animals. Motilily, behaviour and respiration of the animals were normal. Some rats dosed with the bark exuact delivered normaly and no tcratogcnic effect was observed. Postmortum gross examination of the vital organs (heart, lung, kidney, spleen, intestine and liver) did not depict any toxic effect .

The haematological parameters (Hb, WBC, RBC, PCV) were similar in die control and treated rats. Also the plasma level of total protein, albumin, urea, GOT and GPT were similar to the control rats (Table 3).

3.4.3 Subchronic toxicity in Chicks none of the Chicks in all groups (1 -6) died during the experiment. Chicks fed orally with the bark powdcr-dict mixture (2 and 10%) showed no pathological changes and the blood cellular elements and serum constituents (GOT, total protein, albumin, globulin, bilirubin, uric acid, phosphorus and calcium) did not differ from the control (Table 4) .

The Chicks dosed orally with die ethanolic extract (500 mg/kg) showed signs of liver toxicity (fatty vacuoles, dilated and congested sinusoid) without any significant lesions in odicr organs. Injection of the ethanolic extracts to Chicks (50 mg/kg i.p and 10 mg/kg i.m) showed both haematoma in the liver and adhesion between abdominal and thoracic organs. There were no significant changes in the constituents of serum taken from Chicks in all groups . 4. Discussion

The aqueous extract of D.aegyptiaca bark did not affect the activity of the isolated tissues tested. The frecze-drid substance prepared from the bark aqueous extract was also without any effect on these isolated tissues. However larger doses of die substance lowered significantly the contractility and the heart rate of isolated rabbit perfused heart. There is no published infonnation about the pharmacology of D .aegyptiaca bark, but die saponins from B.roxburghii, regarded by Hardnian (1969) as identical to ϋ.eagyptiaca, had no effect on the cardiovascular system of the dog (Bancrji et al., 1981) .

The bark aqueous extracts decreased the serum bilirubin concentration of bile duct-ligated rats in a dose dependant manner. This effect was not shown in non bile duct-ligated Chicks fed with the powdered bark .

A rapid acute toxicity test in rats was carried out using the frecze-dried bark aqueous extract. The extract did not show any sign of toxicity when administered orally up to 2000 mg/kg, and the vital organs were normal. However rats died when injected with ^' doses = 1000 mg/kg. The rats showed nasal bleeding which may be due the hae olytic effects of saponins. The bark, like fruits of die plant, contains saponins (Archibald, 1933; Watt and Breycrs-Brandwijk, 1962; Bashir et al., 1984).

Similar results were obtained when the aqueous bark extracts administered orally and intraperitoneally to mice. The animals tolerated the extracts up to 48 g bark/kg orally and 19.2 g bark/kg inuapctitυneally. The plant seems to be more safer when orally administered than when injected .

The LD50 in mice orally (5440 mg frceze-dried extract/kg), i.p (1320 mg frecze- dried extract kg) were 83.7 and 20.3 limes greater dian the dose used in Sudanese folk medicine to treat jaundice (65 mg frcezc-dried extract/kg; Pcrsoiicl observation) .

The oral administration of he freeze-dricd substance prepared from die bark aqueous extract to rats for 3 weeks was without any significant effect in blood cellular elements or plasma constituents, although doses used were I to 25 times greater than the dose used in Sudanese folk medicine to treat jaundice. The extracts did not induce any tcratogomc effects in new bom pups . Addition of the powdered bark to chick diet for 4 weeks was without effects on body weight gain, blood cellular elements or scrum constituents. However the ethanolic extract of the powdered bark when orally administered showed signs of liver toxicity. Similarly the injection of this extract to chicks (i.p & i.m) induced liver hematoma and adhesion of abdominal & thoracic organs without a significant effect on the serum constituents. Such effects were not observed in rats when the aqueous extract was used orally. The ethanolic extract observed toxicity in chicks may be due to alcohol soluble constituents such as more saponins (Abu-El-Futuh, 1983, 1989; Liu and Nakanishi, 1982) and furanocoumarius (Seida et al., 1981) .

Regarding the toxicity of the aqueous cxuact of the D.aegyptiaca bark we concluded diat the plant is safe when administered orally for a period up to 3 weeks in doses up to 48 g bark kg (= 1920 mg of the frcczcd-dricd substance) .

In conclusion these results point to the significant effectiveness of D.aegyptiaca bark aqueous extract in decreasing scrum bilirubin level in experimental obsUuctive jaundice in rats. Thus it has the potential for treatment of obstructive jaundice in humans . This suggestion is strengthened by the greater safely of the aqueous extract as indicated by experiments in rodents .

Table 1 : The influence of the aqueous extract ofD. aegyptiaca bark on isolated rabbit perfused heart

* I^»<0.05

N = 4

Table 2 : Effect of D.aegyptiaca bark aqueous extracts on serum bilirubin level of bile duct-ligated rats

* P < 0.05

N = 6

Table 3 : Effect of B.aegyptiaca bark aqueous extracts on blood cells and constituents

Treatment Hb PCV WBC RBC Protein Albumin Urea GOT GBΓ «: g100 ml cεll/mms (xlOceil (g100 ml) (g/100 ml) [mg/lOOml) UL UL mms

Control (water) 13 + 0.2 41 + 1.0 4876 ±112 5.71 ±0.94 8.0 ±0.2 4.5 + 0.1 44 ±2-0 70 + 4.0 40 ±1.0

Plant extract 13 ±0.3 42 ±1.0 4974 ±121 5.74 + 1.08- 7.4 ±0.1 4.1+0.1 35 ±2.0 69 ±2.0 34± 1.0 (1625 mg/kg)

N = 10

Table 4 : Effect of B.aegyptiaca Stem bark on Serum Constituents of Chicks

N = 12

MEDICAL OBSERVATION FOLLOWING

TREATMENT WITH A HERBAL MEDICINE IN CASES

OF HIV/AIDS IN THE SUDAN

The incidence rate of AIDS known cases is increasing rapidly in Sudan, five hundred cases were reported in 1998 (AIDS quarter report 1998 ) . The human immunodefcienly virus (HIV) is well known. The virus inters the T. helper cells of the immune system inside the cells it destroys genetic material. The damage is permanent . All body fluids contain T. helper cells, but the concentration is high in bloods, semen and vaginal secretion. There is no simple test to check the virus itself but ELIS A and Western Blot tests are the most common tests wich check the body's reaction to the virus.

As AIDS cases used to come at late stage, clinically they presented by sever weight loss, diarrohea, thrush, itching skin and joint pain. Traditional herbal medicine has been used for management of jaundice and infective hepatitis ( Sayed Osman, from National Center for Research - Khartoum ). Aherbal extract of Balanites aegyptiaca bark ( 30% W/V aqueous extract to be given at 100ml every 8hrs for 30 days for the treatment of acquired immunodeficiency syndrome ( AIDS ) . We started our study about the effectiveness of the above mentioned extract in Khartoum Civil Hospital in 1997. We selected our patients and subjected them to different diagnostic measures and Laboratory investigations and great medical care. We then started giving them the herbalextract three times daily for one month under great medical care and Laboratory investigation for HIV/ AIDS level by use of (EL1SA TEST), some times we gave patients vitamines. The research contiued for one year.

Total number of patients were 20 with age ranging from 2 - 50 years. Male to Female ratio was 4: 1. All ofthem were severe cases. Ten of tliem were selected as cases and ten as control group.

To sum up the results of the study, the cases compared to control obtained very quick clinical improvement in wieght (7 - 10 kg) gained within one month, diarrohea, headache and vomiting were disappeared and movement became normal . the cases were followed for one year ,no relapses occurred. Patients from neighboring countries who received the same treatment did the viral load test in his country where the test is available, the viral load decreased from 500 to 200,0000 when the CD4 : CD8 ratio was low 14 - 16% 75 - 80% after the treatment the viral load became 4. 800 cm3 and CD4 : CD8 is still low. The patient of the control group don't show any improvement till death.

CONCLUSION The herbal extract of Balanitis Egyptiaca which was offered by National Centre for Research - Khartoum with a certificate that it is not toxic to human use in special dose was tried before in 100 patients in Khartoum Hospitals and proved to be harmless, effective, has no side effects or complications ..

We do recommend this extract to be used for the sake of those who suffer from the complications of HIV/ AIDS.

This report consists of three parts. Part one deals with the general introduction about Balaniies aegyptiaca including it's taxonomy, botanical characters, chemical constituents, medicinal folk uses and economic uses. Part two includes some pharmacological and toxicological tests using B-aegypUaca aqueous extract.

The pharmacological tests covered number of isolated tissues (rabbit intestine, rabbit aortic strip, rat uterus, rat stomach strip, rat phrenic-nerve diaphragm preparation and perfused rabbit heart. Toxicity studies covered acute toxicity, lethal dose 50 and subchronic toxicity. Part three deals with clinical results showing the effectiveness of Balaniies aegyptiaca aqueous extract (36% w/v) of Aids age groups (2 - 50 years) of Sudanese patients following treatment 3 times daily for one month.

Also showing its effectiveness in treatment of leukemia patients from 9 -40 years.

MEDICAL OBSERVATION FOLLOWIN TREATMENT WITH A HERBAL MEDICINE IN CASE OF LEUKEMIA

The incidence of Leukemia is more than five cases per 100,000 population and about 75% of new cases are in adult

Untreated Leukemia is a rapidlly killing disease, which charaterized by progressive proliferation and accumulation of abnormal immature blood cells in the bone marrow and other tissues. The progressive disapearence of normal erythrocytes, granulocytes and platelets from the blood leads to fatigability, infection and hemmorageduring the course of disease.

Traditional herbal medicine has been used for centuries in the management of fever, blood animias and jaundice. A herbal extract of Balanites Aegyptiaca bark (30% w/v) aqueous extract to be given 100 ml. 8hrly for one month for the treatment of acute myelocytic Leukemia (AML) in adults and children.

We stated our study about the effectivness of the above-mentioned extract in Radiotherapy Hospital in 1997. We selested our patients and subjected them to different diagnostic measures and stricts laboratory investigations and great medical care.

We started giving them herbal extract three times daily for thirty days under special care and investigations including haemoglobin (HB, total white blood counts "TWBC", ESR, Platelets, Malaria and bone marrow test.

The research continued for one year. The total number of patients were 15 with age range from 9 - 40 and ratio male to female 1 :1

Mild cases were 50%

Sever cases were 50%

We had a control group of 5 patients. To sum up the result of the study, we hereby state that we have obtained very quick treatment for all of 10 patients

Weekly the constant increasing in platelets (25000 weekly), (TWBC500 Weekly).

After one month, blono od differential almost reads normal.

5 - 8 kgs increase in weight, bone marrow complete remission (less than 5% blast cells). One year after treatment no relapse recorded.

The patients of the control group showed gradual improvement due to normal chemotherapy treatment. CONCLUSION

The herbal extract of Balanites Aegyptiaca, which was offered by National Centre for Researches - Khartoum with a certificate that it is not toxic tp human use in special dose was tried in 100 patients in Khartoum Hospital and proved to be harmless, effective, has no side effects or complications and of palatable taste.

We do recommend this extract to be used for the sake of those, who suffer from the complications of Leukemia.

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Claims

CLAIMSBalanites Aegyptiaca (L.) a well known tree in Sudan and other tropical countries for many folk uses. However, it's anti HIV/AIDS or anti Leukemia activity was not discovered before HIV/ AIDS and Leukemia cases started to prevail in Sudan. Our discovery of a plant aqueous extract will treat the patients with HIV/ AIDS and Leukemia. Thus we claim the following:

1. The aqueous extract of the stem bark of B. Aegyptiaca (L.) prepared by boiling 30 gram of the powdered bark in 100 ml. distilled water for five minutes 9 filtration, taken orally as 100 ml every 8 hours for three months treats completely 90% of patients infected with HIV/ AIDS (total of 10). The same dose also 100ml 8hrly for one month is effective in treatment of 100% of patients with Leukemia.

2. The extract prepared as in claim No.l was without any adverse effect in patients suffering from HIV/ AIDS and Leukemia.

3. The therapeutic dose calculated 65-mg/kg bodyweight taken orally of the freeze-dried extract prepared by subjecting the extract in claim No. 1 to freeze-drying.

4. The lethal dose 50 (LD₅0) was calculated as 1320 mg/kg by intraperiotoneal (i.p) and 5440 by oral route in mice.

5. The therapeutic index of the plant extract was calculated as follows:

5440

= 83

65

Thus the extract has a wide range of safety.

6. Finally, we claim our legitimate right for any future utilization, manufacturing or distribution of the aqueous of the stem bark of B. Aegyptiaca or its freeze-dried form in treatment of HIV/ AIDS and Leukemia cases.