WO2011014561A1

WO2011014561A1 - Extracts of medicinal plant and uses thereof

Info

Publication number: WO2011014561A1
Application number: PCT/US2010/043542
Authority: WO
Inventors: Johnny J. He; In-Woo Park; Guangying Chen; Changri Han; Xiaoping Song
Original assignee: He Johnny J; In-Woo Park; Guangying Chen; Changri Han; Xiaoping Song
Priority date: 2009-07-29
Filing date: 2010-07-28
Publication date: 2011-02-03
Also published as: CN102724990A

Abstract

Extracts of traditional Chinese medicinal plants, Annonaceae, Artabotrys pilosus, Annonaceae, Dasymaschalon rostratum, Euphorbiaceae, Trigonostema xyphophylloides, and Dipterocarpaceae, Vatica astrotricha, are described. Pharmaceutical compositions comprising the extracts and treatment of HIV infection is described.

Description

EXTRACTS OF MEDICINAL PLANT AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U. S. C § 119(e) to U.S. Patent Application Serial No. 61/229,426, entitled "EXTRACTS OF MEDICINAL PLANT AND USES THEREOF," filed July 29, 2009. The entirety of the disclosure of the application is incorporated herein by reference.

TECHNICAL FIELD

This invention pertains to compositions comprising extracts of Chinese medicinal plants, processes for preparing extracts of Chinese medicinal plants, and their use in the treatment of HIV infection.

BACKGROUND AND SUMMARY

Highly active anti-retroviral therapy (HAART) is the current HIV/AIDS treatment modality. Despite the fact that HAART is very effective in suppressing HIV-I replication and reducing the mortality of HIV/AIDS patients, it has become increasingly clear that HAART does not offer an ultimate cure to HIV/AIDS. The high cost of the

HAART regimen has impeded its delivery to over 90% of the HIV/ AIDS population in the world. This reality has emphasized the urgent need to develop inexpensive alternative anti-HIV/AIDS therapy. This need has been further manifested by recent clinical trial failures in anti-HIV-1 vaccines and microbicides. Described herein are the anti-HIV activities of a panel of extracts of traditional Chinese medicinal herbal plants, which were obtained from Hainan Island, China, a biologically diverse tropical/subtropical island, and their activities against HIV-I replication.

Human immunodeficiency virus type 1 (HIV-I) causes acquired immune deficiency syndrome (AIDS). At a systemic level, HIV-I primary infection begins with its initial contact with epithelial dendritic cells, followed by its infection of these cells and/or its delivery to CD4+ T cells in lymph nodes. This is followed by rapid virus replication and further dissemination of HIV-I into other lymphoid organs, clinically called viremia, at which HIV-I -infected individuals present a flu-like illness manifested by fever, headache, and others. The severity and duration of the clinical symptoms vary among individuals. Anti-HIV-1 antibodies usually appear 3-6 weeks after infection, while activated CD4+ T cells begin to deplete. Within weeks or months, the number of CD4+ T cells and HIV- 1 viral level in peripheral blood (viral load) stabilize, and a chronic asymptomatic latent infection is established. The latently infected cells will be activated at the late stage of the disease, and the clinical presentations become symptomatic, typically showing fatigue, weight loss, and fever. HIV-I replication and CD4+ T cell depletion accelerate at the symptomatic stage, which makes HIV-I -infected subjects prone to various opportunistic infections, such as Candida, Pneumocystis, toxoplasma, and mycobacteria. CD4+ T cell counts below 200/μl and opportunistic infections are two important characteristics of AIDS patients. Without medical intervention, CD4+ T cell depletion and the immune system destruction continue, eventually leading to death.

CD4+ T lymphocytes are the natural target of HIV-I infection. At the cellular level, HIV-I life cycle begins with binding of HIV-I gpl20 to cellular receptors CD4 and chemokine receptors CCR5 or CXCR4 that are expressed on the surface of HIV- 1 target cells, followed by gp41 conformational change, which in turn leads to virus-cell membrane fusion and entry of the viral core (nucleocapsid) into the cytoplasm. The virion core undergoes uncoating, the viral RNA genome is converted into proviral DNA by the virally encoded enzyme reverse transcriptase (RT). The DNA enters the nucleus and is covalently integrated into the genome of the host cell by the second virally encoded enzyme integrase (IN). The integrated viral DNA serves as the template for viral transcription and synthesis of various components of progeny viruses. Progeny viruses are assembled on and budded through the plasma membrane. As a result, the progeny viruses become encapsulated by a layer of membrane that also harbors the viral envelope glycoproteins. Concomitant with budding, a third virally encoded enzyme protease (PR) processes the core proteins into their final forms, and the virion undergoes a morphologic change known as maturation. This final step primes the progeny viruses for the next round of infection.

In parallel with the progress made in the basic understanding of HIV-I infection and pathogenesis has been the development of anti-HIV-1 therapeutics. The primary targets for anti-HIV- 1 therapeutic development have been two virally encoded enzymes: reverse transcriptase (RT) and HIV protease (PR). The Food and Drug

Administration (FDA) has approved a total of 21 anti-HIV-1 drugs, a majority of these drugs are HIV-I RT and PR inhibitors. Highly active anti-retroviral therapy (HAART), a combination of various anti-HIV-1 drugs effectively suppresses viral replication and has led to a significant reduction in the mortality rate of the disease, increase in the lifespan of HIV/AIDS patients and improvement of the quality of life of these patients. However, issues such as viral reservoirs, drug resistance, high dosages and frequencies, and high cost, have led to a significant crisis in the management of HIV/AIDS patients, particularly in developing nations, where there is the greatest need. It has become evident that HAART does not offer a complete solution to the problem. Meanwhile, relatively fewer anti-HIV-1 therapeutics have been developed to target other steps of HIV-I life cycle including entry, fusion, and integration. On the other hand, recent trials on anti-HIV-1 vaccines and microbicides have shown that some of current vaccine and microbicide strategies not only did not prevent but actually increased HIV-I infection and transmission risks. Therefore, additional and alternative anti-HIV-1 therapeutic strategies are desperately needed to inhibit this virus from destroying the immune system of infected individuals and from spreading to others.

All currently FDA-approved anti-HIV drugs are chemically synthesized.

Development of these drugs involves a long cycle of research, design and optimization, thus these drugs are very expensive. Many of these drugs are structural analogs of host metabolic components; consequently, use of these drugs is often limited by side effects and non-adherence issues. In contrast, medicines of natural origins such as herbs often have a much shorter development cycle and can be relatively inexpensive. Also, the toxicity of nature-derived medications is less often an issue. A recent study has shown that more than two thirds of those who are on anti-HIV medications are also taking alternative therapy, including nearly 25% on those derived from botanicals and Chinese herbs. There is clearly a need to further investigate and develop this alternative anti-HIV therapy, for its contribution to making anti-HIV therapy ultimately affordable and available to all HIV/AIDS-affected individuals including those in developing and underdeveloped countries.

Some efforts have been made to identify natural remedies to combat HIV/AIDS in clinical settings. A number of natural products have been shown to possess anti-HIV-1 activities, including those derived from microorganisms, marine organisms, and plants, and these natural products are believed to inhibit HIV-I replication at various steps of HIV-I life cycle. Currently, more than 150 natural products that have been - A - isolated from marine organisms show promising anti-HIV- 1 activity. One example is cyanovirin-N, an 11 KDa anti-HIV- 1 protein that was initially isolated from the cyanobacterium (blue-green alga) Nostoc ellipsosporum. Cyanovirin-N, it is believed, inhibits HIV-I replication by binding to HIV-I gpl20 and as a result, it inactivates the virus and blocks the fusion the of virus to the cell membrane. This protein is now in Phase II clinical trial for use as an anti-HIV- 1 microbicide.

Traditional Chinese Medicines (TCMs) have a very long history. They have been used to treat various human diseases and are regarded as a state cultural treasure by the Chinese government. Development and standardization of TCMs have recently been proposed as the top biomedical research priority for the next 5-10 years in P. R.

China. Studies on the anti-HIV activities and mechanisms of TCMs are very limited and are expected to accelerate. Currently, HIV-I -inhibitory TCMs are reported to include Scutellaria baicalensis Georgi, Prunella vulgaris, Paeonia Suffruticosa, Rhizoma Polygoni Cuspidati, Radix Notoginseng, Ramulus Visci, and Ajuga Decumbens Thumb.

In one embodiment of the invention, a process for preparing a pharmaceutical composition useful for treating an HIV infection comprising an active fraction, the process comprising the step of obtaining the active fraction from plant material obtained from Euphorbiaceae, Trigonostema xyphophylloides; or

Dipterocarpaceae, Vatica astrotricha; Annonaceae, Artabotrys pilosus; or Annonaceae, Dasymaschalon rostratum is described.

In another embodiment, a process for preparing a pharmaceutical composition useful for treating an HIV infection comprising an active fraction, the process comprising the step of obtaining the active fraction from plant material obtained from Euphorbiaceae, Trigonostema xyphophylloides or Dipterocarpaceae, Vatica astrotricha is described.

In another embodiment, a process for preparing a pharmaceutical composition useful for treating an HIV infection comprising an active fraction, the process comprising the steps of:

a) drying plant material obtained from Euphorbiaceae, Trigonostema xyphophylloides; Dipterocarpaceae, Vatica astrotricha; Annonaceae, Artabotrys pilosus ; or Annonaceae, Dasymaschalon rostratum;

b) grinding the dried plant material; and c) extracting the ground plant material with a first extraction agent to yield a plant extract comprising the active fraction is described.

In another embodiment, the process of the preceding embodiment further comprising the steps of: d) removing substantially all of the first extraction agent to yield a first residue; and e) extracting the first residue with a second extraction agent to yield a second extract comprising the active fraction is described.

a) drying plant material obtained from Euphorbiaceae, Trigonostema xyphophylloides or Dipterocarpaceae, Vatica astrotricha;

b) grinding the dried plant material; and

c) extracting the ground plant material with a first extraction agent to yield a plant extract comprising the active fraction is described.

In another embodiment, a pharmaceutical composition resulting from any one of the preceding process embodiments is described.

In another embodiment, a method of treating a patient in need of relief from an HIV infection, the method comprising the step of administering to the patient a therapeutically effective amount of the pharmaceutical composition described in any of the preceding embodiments is described.

In another embodiment, an active fraction useful for treating HIV infection obtained from plant material comprising one or more plant parts of at least one plant selected from the group consisting of Euphorbiaceae, Trigonostema xyphophylloides;

Dipterocarpaceae, Vatica astrotricha; Annonaceae, Artabotrys pilosus; and Annonaceae, Dasymaschalon rostratum by a process comprising the step of extracting the plant material with a first extraction agent is described.

In another embodiment, a process for preparing an active fraction useful for treating an HIV infection, wherein the process comprises the step of extracting plant material from Euphorbiaceae, Trigonostema xyphophylloides; Dipterocarpaceae, Vatica astrotricha; Annonaceae, Artabotrys pilosus; or Annonaceae, Dasymaschalon rostratum with a first extraction agent is described. In another embodiment, a pharmaceutical composition useful for treating an HIV infection comprising an active fraction wherein the active fraction is prepared by a process comprising the step of extracting plant material from Euphorbiaceae,

Trigonostema xyphophylloides; Dipterocarpaceae, Vatica astrotricha; Annonaceae, Artabotrys pilosus; or Annonaceae, Dasymaschalon rostratum with a first extraction agent.

A method for treating a patient in need of relief from HIV infection, the method comprising the step of administering to the patient a therapeutically effective amount of any of the active fractions or pharmaceutical compositions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE IA. Effects Of The Extracts From TXE And VAD On HIV

Replication. Jurkat cells were infected with HIV-I HXB2 and then exposed to the extracts 24 hr post infection. Fresh extracts were added every other day. Meanwhile, culture supernatants were collected for the RT activity assay. DMSO was the solvent of the extracts and included as a negative control, while AZT was included as a positive control. In addition, Jurkat cells without HIV infection [Ctrl, (-HIV)] and Jurkat cells with HIV infection but without any treatments [Ctrl, (+HIV)] were also included, a) Ctrl ( no HIV infection), b) - Ctrl (HIV infection, no treatment), c) DMSO (1 %), d) DMSO (0.1 %), e) DMSO (0.01%), f) AZT (10 μM), g) AZT (1 μM), h) TXE (100 μg/ml), i) TXE (10 μg/ml), j) TXE (1 μg/ml), k) VAD (100 μg/ml), 1) VAD (10 μg/ml), m) AD (1 μg/ml). These data are representative of three independent experiments.

FIGURE IB. Effects Of The Extracts From TXE And VAD On Cell Survival. Jurkat cells were infected with HIV-I HXB2 and then exposed to the extracts 24 hr post infection. Fresh extracts were added every other day. Aliquots of cells were stained with trypan blue dye and counted for viable cells. DMSO was the solvent of the extracts and included as a negative control, while AZT was included as a positive control. In addition, Jurkat cells without HIV infection [Ctrl, (-HIV)] and Jurkat cells with HIV infection but without any treatments [Ctrl, (+HIV)] were also included, a) Ctrl ( no HIV infection), b) - Ctrl (HIV infection, no treatment), c) DMSO (1 %), d) DMSO (0.1 %), e) DMSO (0.01%), f) AZT (10 μM), g) AZT (1 μM), h) TXE (100 μg/ml), i) TXE (10 μg/ml), j) TXE (1 μg/ml), k) VAD (100 μg/ml), 1) VAD (10 μg/ml), m) AD (1 μg/ml). These data are representative of three independent experiments. FIGURE 2. Effects Of The Extracts On Cell Proliferation And Survival. Jurkat cells were exposed to the extracts for various lengths of time as indicated. Fresh extracts were added every other day. Cells without any treatments, treated with DMSO, or AZT were included as controls. Viable cells were determined using the trypan blue dye staining. These data are representative of three independent experiments.

FIGURE 3. Effect Of The Extracts On Syncytia Formation In HIV-I- Infected Jurkat Cells. Jurkat cells were infected with HIV-I and then exposed to the extracts at 10 μg/mL, 0.1% DMSO, or 5 μM AZT. Syncytia in each of these treatments were counted from 4 random fields from each one of the triplicate samples under a light microscope over the course of 2 weeks infection. The data represented the number of syncytia at day 7 post-infection when the maximal number of syncytia was recorded in the infections receiving no treatments (None) or DMSO. Jurkat cells without HIV-I infection were included as a control (Mock). The data are the mean ± SEM of triplicate

experiments.

FIGURE 4. Direct Effects Of The Extracts On The RT Activity. HIV- 1 virions were assayed for their RT activity in the presence of the extracts at 10 μg/mL. AZT-TP (5 μM) was included as a positive control. DMSO (0.1%) and phosphate- buffered saline (PBS) were included as the solvent control for the extracts and AZT, respectively. The RT reaction without any input HIV-I virions was also included as an assay control. The data are the mean ± SEM of triplicate experiments.

FIGURE 5. Effects Of The Extracts On HIV-I Entry. U87.CD4.CXCR4 cells were treated with TXE (panel A) or VAD (panel B) at a concentration of 10 μg/mL for 30 min and then infected with HIV-Luc viruses pseudotyped T- tropic HIV-I HXB2 envelope (HXB2) or without envelope (-) for 2 hr. Forty-eight hours post infection, cells were harvested for the Luc activity assay. HIV-Luc viruses pseudotyped with VSV-G envelope (VSV-G) were included as a control. DMSO (0.1%) was also included as a solvent control for the extracts. The data are the mean ± SEM of triplicate experiments.

FIGURE 6. Effects Of The Extracts On HIV-I And HIV-I Gene

Expression. Panel A. HIV-Luc viruses pseudotyped T-tropic HIV-I HXB2 envelope (HXB2) were incubated with 10 μg/mL extracts for 2 hr and then used to infect

U87.CD4.CXCR4 cells. Cells were harvested 48 hr for the Luc activity assay 48 hr after infection. Infection with heat-inactivated HIV-Luc/HXB2 viruses (Δ Virus) was included as the control. Panel B. U87.CD4.CXCR4 cells were infected with HIV-Luc viruses pseudotyped T-tropic HIV-I HXB2 envelope (HXB2) or without envelope (-) for 2 hr and then removed of the remaining input viruses by repeated washes with fresh medium. Then, the infected cells were cultured for 48 hr in the presence of the extracts (10 μg/mL) and then harvested for the Luc activity assay. DMSO (0.1%) was also included as a solvent control for the extracts, while 0.5 μM AZT was included as a positive control. The data are the mean ± SEM of triplicate experiments.

FIGURE 7. Effects Of The Extracts On Primary HIV-I Isolate 89.6. Panel A. U87.CD4.CXCR4 and U87.CD4.CCR5 cells were first treated with 10 μg/mL extracts and then infected with HIV-Luc viruses pseudotyped with 89.6 envelope. Infection of HIV-Luc viruses without an envelope was included as the mock infection control. Panel

B. HIV-Luc viruses pseudotyped 89.6 envelope were first incubated with 10 μg/mL extracts and then used to infect U87.CD4.CXCR4 and U87.CD4.CCR5 cells. Infection with heat-inactivated HIV-Luc/89.6 viruses (Δ Virus) was included as the control. Panel

C. U87.CD4.CXCR4 and U87.CD4.CCR5 were first infected with HIV-Luc viruses pseudotyped with 89.6 envelope and then treated with 10 μg/mL extracts or 5 μM AZT. A-C: open bar for U87.CD4.CXCR4 cells; closed bar for U87.CD4.CCR5 cells. Panel D. HIV-Luc viruses pseudotyped with 89.6 envelope were directly treated with 10 μg/mL extracts or 5 μM AZT-TP, the RT activity was determined. The data are the mean ± SEM of triplicate experiments.

FIGURE 8. Anti-HIV Components Of TXE And VAD Extracts. Jurkat cells were infected with HIV-I HXB2 and then exposed to 10 μg/mL TXE (panel A), VAD (panel B), or each of its partition subtractions from petroleum ether (PE), chloroform (CF), ethyl acetate (EA) and n-butanol (BT) 24 hr post infection. Fresh extracts or subfraction were added every other day. Meanwhile, culture supernatants were collected for the RT activity assay, and aliquots of cells were stained with trypan blue dye and counted for viable cells. DMSO was the solvent of the extracts and subfractions and included as a vehicle control. The RT data from the supernatants collected at day 9 of the peak viral replication were presented. Extracts and their subfractions showed no apparent cytotoxic effects on the cells. The data are the mean ± SEM of triplicate experiments. FIGURE 9. Effects of extracts on HIV-I replication. One million Jurkat cells were infected with HIV-I HXB2 corresponding to a 10,000 cpm RT activity for 24 hr and then treated with 10 μg/mL fresh extracts from the leaves, stems and roots of Annonaceae, Artabotrys pilosus; a) AAP-leave extract (AAP-I), b) AAP-stem extract (AAP-s), and c) AAP-root extract (AAP-r); and the roots of Annonaceae, Dasymaschalon rostratum; d) ADR-roots (ADR-r) extract; every other day. The cell culture supernatants were collected at indicated time points for the RT activity assay. All extracts were dissolved in DMSO, and DMSO was used as a vehicle control (e). Mock-infected Jurkat cells (g) and HIV-infected cells without any treatments were also included as controls (f). These data are representative of three independent experiments.

FIGURE 10. Effects of extracts a) AAP-I, b) AAP-s, c) AAP-r, d) ADR-r, e), and control, DMSO on Jurkat cell survival. Jurkat cells were infected and treated as in FIGURE 9. At each time point, an aliquot of cells was collected, stained with Trypan blue dye, and then counted for the cell viability. These data were representative of three independent experiments. Entry f) represents mock infected cells with no treatment and entry g) represents uninfected cells.

FIGURE 11. Effects of extracts on the HIV-I long terminal repeat (LTR) promoter activity. CD4+ T lymphocytes CEM stably expressing green fluorescence protein (GFP) under the control of HIV-I LTR promoter were treated with 10 μg/mL each of the extracts for 3 days (Panel A) and 7 days (Panel B). Then, the cells were collected for measurement of GFP expression by flow cytometry. The data are the geometric means of the GFP expression level and are the mean ± SEM of triplicate experiments.

FIGURE 12. Effects of extracts on the HIV-I RT activity. Purified HIV-I virions were incubated with 10 μg/mL each of the extracts for the RT activity assay. The RT inhibitor AZT (5 μM) was included as a positive control, while DMSO was used as the vehicle control for the extracts. The sample without any treatments (mock) was also included.

FIGURE 13. Effects of extracts on HIV-I entry. Panel A.

U87.CD4.CXCR4 cells were treated with 10 μg/mL each of the extracts for 1 hr and then infected with HIV-Luc viruses pseudotyped T-tropic HIV-I HXB2 envelope. Panel B. Similar experiments were performed with U87.CD4.CCR5 cells and HIV-Luc viruses pseudotyped M-tropic HIV-I YU-2 envelope. HIV-Luc viruses pseudotyped with VSV-G envelope and no envelope were included as the positive and negative infection controls, respectively (data not shown). The data are the mean ± SEM of triplicate experiments.

FIGURE 14. Effects of extracts on HIV-I infectivity. HIV-Luc virus pseudotyped with HXB2 (Panel A) or YU-2 envelope (Panel B) was first incubated with 10 μg/mL each of the extracts at 37⁰C for 2 hr. The virus was recovered by centrifugation and then used to infect either U87.CD4.CXCR4 (for HXB2) or U87.CD4.CCR5 cells (for YU-2). The cells were harvested for the luciferase reporter gene assay 48 hr post infection. The data are the mean ± SEM of triplicate experiments.

FIGURE 15. Effects of extracts on HIV-I post-entry. U87.CD4.CXCR4 and U87.CD4.CCR5 cells were first infected with HIV-Luc virus pseudotyped with HXB (Panel A) or YU-2 envelope (Panel B). Following medium change, the cells were treated with 10 μg/mL each of the extracts for 48 hr and then harvested for the luciferase reporter gene assay. The data are the mean ± SEM of triplicate experiments.

DETAILED DESCRIPTION

Traditional Chinese Medicine (TCM) dates back to 2000 to 3000 years.

Medicinal herbs are a major component of TCM. It is estimated that over 600 different herbs have been used to treat various human diseases including those caused by virus infection. Hainan Island, the second largest island off the coast of China, is located in the South China Sea and in the tropics at about 18° N latitude. There are about 4,200 plant species, 630 of which are listed as endemic to the island and some are nearly extinct.

Described herein is the selection of 12 medicinal herbal plants that have been used to treat various human diseases by local ethnic Chinese in Hainan Island, China (Table 1), extraction of these plants with ethanol, and testing of the extracts for anti-HIV activity. CD4+ T lymphocytes Jurkat were infected with a replication-competent T-tropic HIV- 1 strain HXB2 and then HIV- 1 replication was monitored over a course of 2 weeks in the presence of the plant extracts at concentrations of 1, 10, 100 μg/mL. The solvent of the extracts, DMSO, and an HIV-I RT inhibitor AZT were included in these experiments. This initial testing was repeated three times. Compared to the untreated control or DMSO treatment, 6 of these 17 extracts: from the stem of Euphorbiaceae, Trigonostema xyphophylloides (TXE), from the stem of Dipterocarpaceae, Vatica astrotricha (VAD), from the leaf of Annonaceae, Artabotrys pilosus (AAP-I), from the stem of Annonaceae, Artabotrys pilosus (AAP-s), from the root Annonaceae, Artabotrys pilosus (AAP-r), and from the root of Annonaceae, Dasymaschalon rostratum (ADR-r) displayed inhibition of HIV- 1 replication at 10 μg/mL or higher at day 9 post infection (dpi) (FIGURES IA and 10). The treatment control AZT inhibited HIV-I replication. Extracts VAD and TXE at 1 μg/mL showed little effect on HIV-I replication but dose-dependent anti-HIV activity at 10 μg/mL or higher. Cell survival of all treatments was monitored throughout the experiments by trypan blue dye staining. Compared to the untreated control, both TXE and VAD treatments showed cell growth kinetics similar to that of DMSO-treated cells and of the no-treatment controls (FIGURE IB). The decline in the number of viable cells in HIV-I -infected cells likely results from the infection-induced cell death, as the cell number began to recover from AZT treatment toward the end of the treatment. These results provide initial evidence that TXE and VAD are inhibitory to HIV-I replication.

Extracts from Euphorbiaceae, Trigonostema xyphophylloides (TXE) and Dipterocarpaceae, Vatica astrotricha (VAD) inhibited HIV-I replication and syncytia formation in CD4+ T cells, and had little adverse effects on host cell proliferation and survival. TXE and VAD did not show any direct inhibitory effects on the HIV-I RT enzymatic activity. To determine the inhibitory mechanisms, a single-round HIV- luciferase reporter virus system (HIV-Luc) pseudo-typed with T tropic or M tropic HIV-I envelope was used to infect target cells. Pre-treatment of viruses with these extracts had little effect on the infectivity of HIV-Luc viruses, and treatment of HIV-Luc-infected cells with these extracts did not lead to any significant changes in the Luc gene expression. Without being bound by theory, it is believed that these results suggest that these extracts block HIV-I replication by blocking HIV-I interaction with target cells, i.e., the interaction between gpl20 and CD4/CCR5 or gpl20 and CD4/CXCR4.

To ascertain and establish non-toxic working concentrations of the extracts, the effects of these extracts on cell survival and growth kinetics in the absence of HIV-I infection were measured. Jurkat cells were treated with TXE or VAD at 1, 10 and 100 μg/mL and fresh extract was added every other day and cell survival and growth monitored. DMSO and AZT were included as controls in these experiments. Jurkat cells without any treatment were also included as a control. The cell survival and growth kinetics appeared to be indistinguishable among the cells receiving no treatment, AZT (10 μM), and 10 μg/mL TXE and VAD and its corresponding DMSO concentration, i.e., 0.1% (FIGURE 2). Similar results were obtained between TXE and VAD at 1 and 100 μg/mL and their respective solvent DMSO concentrations 1% and 0.01% (data not shown), suggesting that TXE and VAD are not toxic at a concentration up to 100 μg/mL. Based on these results, the concentration of 10 μg/mL for TXE and VAD extracts was chosen for all following mechanistic studies.

Productive HIV- 1 infection of CD4+ T cells in vitro is characterized by formation of multinucleated giant cells, so-called syncytia, which likely results in CD4+ T cell depletion in HIV-I -infected subjects. The effects of these two extracts on syncytia formation are described. Jurkat cells were infected, treated with 10 μg/mL TXE or VAD, and monitored for syncytia formation over a course of 2 weeks. AZT (5 μM) and DMSO (0.1%) treatments were included as controls. Uninfected and HIV-I -infected Jurkat cells were also present, as controls. The number of syncytia reached the highest value at day 7 post infection. Compared to untreated and DMSO-treated HIV-I -infected Jurkat cells, TXE, VAD and AZT treatments all showed reduction in the number of syncytia (FIGURE 3). There were few syncytia in Jurkat cells that received no HIV-I -infection. These results are in agreement with the inhibitory effects of these extracts on HIV- 1 replication (FIGURE IA).

HIV- l is a member of the retrovirus family. An important feature of these viruses is that replication of these viruses involves the conversion of their RNA viral genome to proviral DNA, which is catalyzed by a unique virally encoded enzyme called reverse transcriptase (RT). Herein described is a comparison of the RT enzymatic activity of HIV-I virions in the presence and absence of the extracts. HIV-I virions were lysed to release the RT and the RT activity assay was performed in the presence of TXE and VAD (10 μg/mL). The RT inhibitor AZT (5 μM) was included as a control in these

experiments. In addition, PBS and DMSO (0.1%) were included as the solvent controls for AZT and TXE and VAD, respectively. The RT reaction without any HIV-I virions was used as an assay blank control. As expected, AZT potently inhibited HIV-I RT activity. However, inclusion of TXE and VAD in the RT reaction did not show any significant difference in RT activity from the PBS and DMSO control (FIGURE 4).

Without being bound by theory, it is believed that these results indicate that TXE and

VAD-induced inhibition of HIV-I replication is not due to their effects on the RT activity but on other steps of HIV life cycle. Leaf extracts from AAP (AAP-I) and stem extracts from AAP (AAP-s) showed some inhibition of RT activity (FIGURE 12)

HIV-I infection begins with HIV-I envelope gpl20 binding to CD4 and chemokine co-receptors CCR5 (for M-tropic strains) or CXCR4 (for T-tropic strains) on the cell surface of HIV-I target cells. Herein described are the effects that the extracts had on HIV-I entry into cells. The replication-defective HIV-Luc reporter system has the HIV- 1 env gene inactivated and the firefly luciferase (Luc) gene in place of HIV- 1 nef . Such a design allows in trans complementation of any viral envelope proteins including HIV- 1 envelope proteins and one single round viral infection to accurately determine HIV-I entry by the sensitive Luc activity assay. HIV-Luc reporter viruses pseudotyped with T-tropic HIV- 1 HXB2 envelope were prepared. To determine effects of these extracts on HIV-I entry, U87.CD4.CXCR4 cells were pre-incubate with these extracts at 10 μg/mL and then these cells were infected with these viruses. The Luc activity of these cells was measured. HIV-Luc reporter viruses pseudotyped with vascular stomatitis virus envelope glycoprotein (VSV-G) or HIV-Luc viruses without any viral envelopes, which were positive and negative controls, respectively, were also prepared in these experiments. DMSO (0.1%) was included as the solvent control for these extracts. Compared to the DMSO control, pre-incubation of U87.CD4.CXCR4 with TXE almost completely blocked infection of HIV-Luc pseudotyped with HIV-I HXB2 envelope but had no effects on that of HIV-Luc pseudotyped with VSV-G (FIGURE 5, panel A). Similar results were obtained with VAD extract (FIGURE 5, panel B). Extracts AAP-I, AAP-s, AAP-r, and ADR-r showed inhibition of infection of U87.CD4.CXCR4 cells by HIV-Luc pseudotyped with HIV-I HXB2 envelope (FIGURE 13, panel A) and inhibition of infection of

U87.CD4.CCR5 cells with HIV-Luc viruses pseudotyped M-tropic HIV-I YU-2 envelope. Without being bound by theory, these results suggest that AAP-I, AAP-s, AAP-r, ADR-r, TXE, and VAD inhibit HIV-I replication by blocking HIV-I entry into target cells.

Described herein is a modified experimental scheme using the same replication-defective single round HIV-Luc reporter system to further ascertain that TXE and VAD inhibit HIV-I replication at the entry step, to determine whether TXE and VAD directly inactivate HIV-I, or had any effects at other steps of HIV-I life cycle. The same amount of HIV-Luc viruses pseudotyped with HXB2 envelope were first incubated in 10 μg/mL TXE, VAD, or 0.1% DMSO at 37 ⁰C for 2 hr. The viruses were recovered by centrifugation and used to infect U87.CD4.CXCR4 cells. The cells were cultured for 48 hr before being harvested for the Luc activity assay. The Luc activity showed no significant difference in the viral infectivity between DMSO treatment control and TXE or VAD treatment, while heat- inactivated viruses had little infectivity (FIGURE 6, panel A). U87.CD4.CXCR4 cells were infected with HIV-Luc viruses pseudotyped with HIV-I

HXB2 envelope at 37 ⁰C for 2 hr. The remaining input viruses were removed by repeated washes with fresh medium and these cells were cultured for 48 hr in the presence of these TXE or VAD extracts (10 μg/mL) or 5 μM AZT. The cells were assayed for the Luc activity assay. Compared to the AZT control, TXE and VAD treatment did not show any differences in the Luc activity of HIV-infected cells from the DMSO treatment (FIGURE 6, panel B). Without being bound by theory, it is believed that taken together, these results show that the observed inhibition of TRX and VAD on HIV-I replication is not due to inactivation of HIV-I by these extracts or the blockage at the post-entry step, further supporting the possibility that TRE and VAD inhibit HIV-I replication at the entry step of the viral life cycle. Incubation of HIV-Luc virus pseudotyped with HXB2 or HIV-luc virus pseudotyped with YU-2 with AAP-I, AAP-s, AAP-r, or ADR-r did not inhibit the ability of the treated viruses to infect U87.CD4.CXCR4 or U87.CD4.CCR5 cells, respectively (FIGURE 14, panel A and panel B).

Herein described are similar experiments conducted with the dual tropic primary HIV-I isolate 89.6 [Doranz BJ, et al, Cell 1996, 85:1149-1158] to investigate whether TXE and VAD were also capable of blocking this virus from entering into its target cells. U87.CD4.CXCR4 and U87.CD4.CCR5 cells were treated with 10 μg/mL of TXE or VAD, and then the cells were infected with HIV-Luc viruses pseudotyped with HIV-I 89.6 envelope. Compared to the DMSO treatment control, both TXE and VAD treatment showed significant lower Luc activities in both U87.CD4.CXCR4 and

U87.CD4.CCR5 cells (FIGURE 7, panel A), indicating that they blocked HIV-I 89.6 infection. The effects of the extracts on HIV-I 89.6 itself and at the post-entry step were determined as described. Similarly to HXB2 viruses, 89.6 viruses did not show any changes in their infectivity when they were exposed to the extracts prior to the infection (FIGURE 7, panel B). Also, 89.6 viruses did not show any changes in their post-entry replication when 89.6 infection occurred prior to the extract treatment (FIGURE 7, panel C). Furthermore, these extracts did not have any direct inhibitory effects on the RT enzymatic activity of the HIV-I 89.6 viruses (FIGURE 7, panel D). Without being bound by theory, it is believed that these data demonstrate that TXE and VAD both possess the similar activity of blocking entry of primary HIV-I isolate 89.6.

These two extracts, were next partitioned into four subtractions using organic solvents of different hydrophobicity and polarity, that is, petroleum ether (PE), chloroform (CF), ethyl acetate (EA) and n-butanol (BT). Described herein are the effects of these subfractions on HIV-I replication. As showed above, TXE treatment inhibited HIV-I replication in a significant fashion (FIGURE 8, Panel A). Compared to the DMSO- treated control, HIV replication was lower in cells treated with CF and BU subfractions, while HIV replication showed little changes in cells treated with its PE and EA

subfractions. In contrast, the PE, CF, and EA subfractions of the VAD extract had lower HIV-I replication than the DMSO control, while its BT subtraction displayed no anti-HIV activity (FIGURE 8, panel B). Similar results were obtained from the single-round infection assay (data not shown). These results suggest that the active anti-HIV

components can be further isolated from both TXE and VAD extracts and may differ between these two extracts.

Extracts of traditional Chinese medicinal herbal plants were screened for their anti-HIV activities using a well-established HIV-I replication system (Table 1). Extracts from the stem of Euphorbiaceae, Trigonostema xyphophylloides (TXE) and the stem of Dipterocarpaceae, Vatica astrotricha (VAD) inhibited HIV-I replication without apparent effects on cell proliferation and cell survival (FIGURE 1 and FIGURE X). The inhibitory effects of these two extracts were further corroborated by the finding that these extracts prevented HIV-infected cells from forming syncytia (FIGURE 3). No effects of these extracts on HIV-I RT enzymatic activity was detected (FIGURE 4). Without being bound by theory, it is believed that the observations described herein suggest that these extracts potently blocked HIV-I from entering its target cells (FIGURE 5). These extracts had little effects on post-entry HIV-I gene expression (FIGURE 6). Similar results were obtained with the primary isolate, HIV-I 89.6 which displays dual tropism, using both CXCR4 and CCR5 for entry into cells (FIGURE 7). Taken together, these studies revealed the anti-HIV activities of these two plant extracts and suggest that their anti-HIV activity results from interfering with HIV-I entry. AAP-I, AAP-s, AAP-r, ADR-r, TXE, and VAD extracts possess potent inhibitory activities against HIV-I replication and entry of both T and M tropic HIV-I isolates.

In one embodiment of the invention, a process for preparing a pharmaceutical composition useful for treating an HIV infection comprising an active fraction, the process comprising the step of obtaining the active fraction from plant material obtained from Euphorbiaceae, Trigonostema xyphophylloides or

Dipterocarpaceae, Vatica astrotricha.

In another embodiment of the invention, a process for preparing a pharmaceutical composition useful for treating an HIV infection comprising an active fraction, the process comprising the step of obtaining the active fraction from plant material obtained from Euphorbiaceae, Trigonostema xyphophylloides; or

Dipterocarpaceae, Vatica astrotricha; or Annonaceae, Artabotrys pilosus ; or Annonaceae, Dasymaschalon rostratum.

b) grinding the dried plant material; and

c) extracting the ground plant material with a first extraction agent to yield a plant extract comprising the active fraction is described. In another embodiment, a process for preparing a pharmaceutical composition useful for treating an HIV infection comprising an active fraction, the process comprising the steps of:

a) drying plant material obtained from Euphorbiaceae, Trigonostema xyphophylloides; or Dipterocarpaceae, Vatica astrotricha; or Annonaceae, Artabotrys pilosus; or Annonaceae, Dasymaschalon rostratum;

b) grinding the dried plant material; and

c) extracting the ground plant material with a first extraction agent to yield a plant extract comprising the active fraction is described. In another embodiment, the process described in either of the preceding embodiments wherein the plant is

Euphorbiaceae, Trigonostema xyphophylloides is described. In another embodiment, the processes described above, wherein the plant is Dipterocarpaceae, Vatica astro tricha are described. In another embodiment, the processes described above, wherein the plant is Annonaceae, Artabotrys pilosus is described. In another embodiment, the processes described above, wherein the plant is Annonaceae, Dasymaschalon rostratum is described. In another embodiment, the plant material is leaf material. In another embodiment, the plant material is stem material. In an alternative embodiment, the plant material is root material. In yet another embodiment, the process of any one of the preceding

embodiments wherein the first extraction agent is a polar or a non-polar solvent, or a mixture thereof is described. In another embodiment, the process of any one of the preceding embodiments wherein the first extraction agent is a mixture of ethanol and water is described. In some illustrative embodiments, the mixture is about 70% to about 80% ethanol about 30% to about 20% water or the mixture is about 75% ethanol to about 25% water.

In another embodiment, the process of any one of the preceding

embodiments wherein the extraction is performed at about 8O⁰C is described.

In another embodiment, the process of any one of the preceding

embodiments further comprising the steps of: d) removing substantially all of the first extraction agent to yield a first residue; and e) extracting the first residue with a second extraction agent to yield a second extract comprising the active fraction is described.

In another embodiment, the process of the preceding embodiment wherein the second extraction agent is a polar, a non-polar solvent or a combination thereof, i.e. petroleum ether, methylene chloride, ethyl acetate, n-butanol, or a combination thereof, or the like, is described.

In another embodiment, a pharmaceutical composition resulting from the process of any one of the preceding embodiments is described. In another embodiment, the pharmaceutical composition of the preceding embodiment further comprising a pharmaceutically acceptable carrier, excipient, diluent, or combination thereof is described.

In another embodiment, a method of treating a patient in need of relief from an HIV infection, the method comprising to the patient a therapeutically effective amount of the pharmaceutical composition described in one of the preceding embodiments is described. In another embodiment, a method of preventing an HIV infection comprising the step of administering the pharmaceutical composition described in any one of the preceding embodiments is described

Embodiments of the invention are also described in the following clauses. 1. An active fraction useful for treating HIV infection obtained from plant material comprising one or more plant parts of at least one plant selected from the group consisting of Euphorbiaceae, Trigonostema xyphophylloides, Dipterocarpaceae, Vatica astrotricha, Annonaceae, Artabotrys pilosus, and Annonaceae, Dasymaschalon rostratum by a process comprising the step of extracting the plant material with a first extraction agent..

2. The active fraction of clause 1 wherein the process further comprises the steps of drying the plant material and grinding the plant material, wherein the drying step precedes the extracting step.

3. The active fraction of clause 1 or 2 wherein the first extraction agent comprises a polar or a non-polar solvent, or a mixture thereof.

4. The active fraction of any one of clauses 1 to 3 wherein the first extraction agent comprises a mixture of ethanol and water.

5. The active fraction of any one of clauses 1 to 4 wherein the extraction agent comprises a mixture of from 70% to 95% ethanol and from 30% to 5% water.

6. The active fraction of any one of clauses 1 to 5 wherein the extraction is performed at a temperature of from 75° C to 85⁰C.

7. The active fraction of any one of clauses 1 to 6 in the form of a concentrate wherein the process further comprises the step of removing the first extraction agent by evaporation.

8. A second active fraction obtained by a process comprising the step of extracting the concentrate of any one of clauses 1 to 7 with a second extraction agent.

9. The second active fraction of any one of clauses 1 to 8 wherein the second extraction agent is a polar solvent, a non-polar solvent, or a combination thereof.

10. The second active fraction of any one of clauses 1 to 9 wherein the second extraction agent is petroleum ether, methylene chloride, ethyl acetate, n-butanol, or a combination thereof. 11. The active fraction of any one of clauses 1 to 10 wherein the plant is Euphorbiaceae, Trigonostema xyphophylloides.

12. The active fraction of any one of clauses 1 to 11 wherein the plant is Dipterocarpaceae, Vatica astrotricha.

13. The active fraction of any one of clauses 1 to 12 wherein the plant is

Annonaceae, Artabotrys pilosus.

14. The active fraction of any one of clauses 1 to 13 wherein the plant is Annonaceae, Dasymaschalon rostratum.

15. The active fraction of any one of clauses 1 to 14 wherein the plant part is root.

16. The active fraction of any one of clauses 1 to 15 wherein the plant part is stem.

17. The active fraction of any one of clauses 1 to 16 wherein the plant part is leaf.

18. A process for preparing an active fraction useful for treating an HIV infection, wherein the process comprises the step of extracting plant material from

Euphorbiaceae, Trigonostema xyphophylloides, Dipterocarpaceae, Vatica astrotricha, Annonaceae, Artabotrys pilosus, or Annonaceae, Dasymaschalon rostratum with a first extraction agent.

19. The process of clause 18 wherein the process further comprises the step of drying the plant material, wherein the drying step precedes the extracting step.

20. The process of clause 18 or 19 wherein the process further comprises the step of grinding the plant material.

21. The process of any one of the clauses 18 to 20 wherein the first extraction agent comprises a polar or a non-polar solvent, or a mixture thereof.

22. The process of any one of the clauses 18 to 21 wherein the first extraction agent comprises a mixture of ethanol and water.

23. The process of of any one of the clauses 18 to 22 wherein the extraction agent comprises a mixture of from 70% to 95% ethanol and from 30% to 5% water.

24. The process of any one of the clauses 18 to 23 wherein the extraction is performed at a temperature of from 75° C to 85⁰C. 25. The process of any one of the clauses 18 to 24 wherein the process further comprises the step of removing the first extraction agent by evaporation to yield a concentrate.

26. The process of any one of the clauses 18 to 25 wherein the process further comprises the step extracting the concentrate with a second extraction agent.

27. The process of any one of the clauses 18 to 26 wherein the second extraction agent is a polar solvent, a non-polar solvent, or a combination thereof.

28. The process of any one of the clauses 18 to 27 wherein the second extraction agent is petroleum ether, methylene chloride, ethyl acetate, n-butanol, or a combination thereof.

29. The process of any one of the clauses 18 to 28 wherein the plant material is from Euphorbiaceae, Trigonostema xyphophylloides.

30. The process of any one of the clauses 18 to 29 wherein the plant material is from Dipterocarpaceae, Vatica astrotricha.

31. The process of any one of the clauses 18 to 30 wherein the plant material is from Annonaceae, Artabotrys pilosus.

32. The process of any one of the clauses 18 to 31 wherein the plant material is from Annonaceae, Dasymaschalon rostratum.

33. The process of any one of the clauses 18 to 32 wherein the plant material comprises a part of the plant selected from the group consisting of root, stem, leaf, seeds, and flowers.

34. A pharmaceutical composition useful for treating an HIV infection comprising an active fraction wherein the active fraction is prepared by a process comprising the step of extracting plant material from Euphorbiaceae, Trigonostema xyphophylloides, Dipterocarpaceae, Vatica astrotricha, Annonaceae, Artabotrys pilosus, or Annonaceae, Dasymaschalon rostratum with a first extraction agent.

35. The composition of clause 34 wherein the process further comprises the step of drying the plant material wherein the drying step precedes the extracting step.

36. The compostion of clause 34 or 35 wherein the process further comprises the step of grinding the plant material.

37. The composition of any one of the clauses 34 to 36 wherein the first extraction agent comprises a polar or a non-polar solvent, or a mixture thereof. 38. The composition of any one of the clauses 34 to 37 wherein the first extraction agent comprises a mixture of ethanol and water.

39. The composition of any one of the clauses 34 to 38 wherein the extraction agent comprises a mixture of from 70% to 95% ethanol and from 30% to 5% water.

40. The composition of any one of the clauses 34 to 39 wherein the extraction is performed at a temperature of from 75° C to 85⁰C.

41. The composition of any one of the clauses 34 to 40 wherein the process further comprises the step of removing the first extraction agent by evaporation to yield a concentrate.

42. The composition of any one of the clauses 34 to 41 wherein the process further comprises the step extracting the concentrate with a second extraction agent.

43. The composition of any one of the clauses 34 to 42 wherein the second extraction agent is a polar solvent, a non-polar solvent, or a combination thereof.

44. The composition of any one of the clauses 34 to 43 wherein the second extraction agent is petroleum ether, methylene chloride, ethyl acetate, n-butanol, or a combination thereof.

45. The composition of any one of clauses 34 to 44 wherein the plant material is from Dipterocarpaceae, Vatica astrotricha.

46. The composition of any one of clauses 34 to 45 wherein the plant material is from Annonaceae, Artabotrys pilosus.

47. The composition of any one of clauses 34 to 46 wherein the plant material is from Annonaceae, Dasymaschalon rostratum.

48. The composition of any one of clauses 34 to 47 wherein the plant material comprises a part of the plant selected from the group consisting of roots, stems, leaves, seeds, and flowers.

49. A method for treating a patient in need of relief from HIV infection, the method comprising the step of administering to the patient a therapeutically effective amount of the active fraction of any one of the preceding clauses or the pharmaceutical composition of any one of the preceding clauses . In alternative embodiments, the active fractions may be formulated for oral, rectal, vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intrathecal), and oral administration.

As used herein, the term "active fraction" generally refers to a fraction or an extract comprising a pharmacologically active agent, whether a component, a combination of components, a biological metabolite, a derivative thereof or a combination of the above, that exhibits anti-HIV activity. It is appreciated that the anti-HIV activity may be due to a single component, a combination of components, or biologic metabolites or derivatives thereof.

It is appreciated that when preparing an extract from a part of the plant, e.g. stems, leaves, roots, flowers, seeds, and the like, some amount of plant material from one or more other parts of the plant may be mixed in with the targeted part of the plant. It is appreciated that before obtaining an active fraction from a plant one or more of the following steps may occur: growing the plants, harvesting the plants or collecting the plants or collecting a portion of the plants, or cleaning the plant material to remove foreign material.

It is appreciated that the step of grinding the plant material may occur prior to the extraction step or during the extraction step.

As used herein, the term "removing substantially all" generally refers to removing not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%, or not less than 99.5% of the material removed.

In another embodiment, conventional methods of producing extracts based upon treatment of plant material with an extracting agent to obtain a raw or primary extract which after an optional treatment for removal of fines, e. g. by sedimentation or filtration, contains the extracting agent and the plant constituents that are soluble in the extracting agent are described. In another embodiment, extraction agents comprising polar or non-polar solvents, or a combination thereof are described. Illustrative examples of solvents useful as extraction agents are water, alcohols, e.g. methanol, ethanol, n-propanol, iso-propanol, n-butanol, sec-butanol, iso-butanol, t-butanol, and the like, acetonitrile, ethyl acetate, propyl acetate, n-butyl acetate, iso-propyl acetate, and the like, pentane, petroleum ether, hexanes, heptane, toluene, methylene chloride, chloroform, acetic acid, tetrahydrofuran, ethyl ether, t-butyl methyl ether, and the like, or combinations thereof. In another embodiment, the extraction agent comprises a mixture of an alcohol and water.

The term "extract" as used herein without further specification generally refers to any form of the product of extraction with or without the extracting agent and regardless of the physical form (i. e. viscous, pasty or solid). The extractions described herein can be carried out at any temperature range from about -3O⁰C to about the boiling point of the extraction agent. It is appreciated that the extraction can be preformed at a modified pressure to raise or lower the boiling point of the extraction agent.

In another embodiment, the primary extract is then concentrated by partial evaporation of the extracting agent so as to remove its more volatile components to form what is called a concentrated extract, typically containing 5-50 % by volume, of residual solvent, e.g.. water. Upon further removal of solvent, a solid, pasty, or liquid material is obtained that is substantially free of the solvent used for extraction of the plant material. This product (also termed "extract" or "active fraction") can then be used as such or processed to produce specific application forms, optionally adding adjuvants, additives, coating components, diluents, excipients, and the like.

The active fractions described herein may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intraurethral, intrasternal, intramuscular, and subcutaneous delivery. Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile nonaqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. The solubility of an active fraction used in the preparation of a parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.

Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release formulations. Thus, an active fraction may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active fraction. Examples of such formulations include drug-coated stents and poly(dl-lactic- coglycolic)acid (PGLA) microspheres.

The pharmaceutical compositions containing the active fraction may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such

compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparation.

Formulations for oral use include tablets which contain the active fraction in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium chloride, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, or alginic acid; binding agents, for example, starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay

disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active fraction is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules wherein the active fraction is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions usually contain the active materials in admixture with appropriate excipients. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally- occurring phosphatide, for example, lecithin; a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example,

heptadecaethyleneoxycetanol; a condensation product of ethylene oxide with a partial ester derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate; or a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example, polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, n-propyl, or p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active fraction in a vegetable oil, for example, arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin, or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active fraction in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring, and coloring agents, may also be present.

The pharmaceutical compositions of the active fractions described herein may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oils, or a mineral oil, for example, liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soybean lecithin; and esters including partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan mono-oleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol, or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring agents, and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid also find use in the preparation of injectibles.

The active fractions described herein may also be administered in the form of a suppository, pessary, or enema for rectal or vaginal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal or vaginal temperature and will therefore melt to release the drug, for example, cocoa butter, and polyethylene glycols. Formulations for rectal/vaginal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release formulations.

As used herein the term "treatment" includes curative, palliative and prophylactic treatment.

It is also appreciated that in the foregoing embodiments, certain aspects of the invention are presented in the alternative. One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Accordingly, for all purposes, the present invention encompasses not only the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

EXAMPLES AND METHODS

EXAMPLE 1

Preparation of plant extracts. All plants were collected at national tropical forest parks in Hainan Island, the People's Republic of China (P. R. China) including

Jianfengling, Bawangling, or Mt. Diaoluo (Table 1). Scientific names and classification of these plants were validated by Prof. Qiongxin Zhong, a plant taxonomist at Department of Biology Hainan Normal University, Haikou, P. R. China. Samples of these plants were kept at the Hainan Provincial Key Laboratory of Tropical Pharmaceutical Herbal

Chemistry, Haikou, P. R. China. Plant samples were first air dried, ground, and continued to be dried in a pressurized oven at 40 ⁰C and 0.08 MPa. The dried and ground materials were then subjected to 3 rounds of refluxing extraction in 75% ethanol at 80 ⁰C. The ethanol extracts were then concentrated to become ointment in a revolving depressurized vacuum evaporator at 55 ⁰C. The ointment was further lyophilized to the final form of powder and stored in a desiccator. The powders were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL with gentle shaking overnight at room temperature. Then, the mixtures were removed of any remaining undissolved substances by centrifugation at 3,000 rpm for 10 min followed by filtration through a 0.4 μm syringe filter. The cleared ones were used in all experiments.

Table 1. Tropical plants selected to make extracts for anti-HIV screening

EXAMPLE 2

Cells, HIV-I HXBc2 viruses, and chemicals. Jurkat cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and lOOμg/mL streptomycin sulfate. U87.CD4.CXCR4 and U87.CD4.CCR5 cells expressing CD4/CXCR4 and CD4/CCR5 respectively were obtained from the NIH AIDS Reagent Program [Bjorndal A, et al., J Virol 1997, 71:7478-7487; the disclosure of the foregoing is incorporated herein in its entirety by reference. In addition, the entirety of the disclosure of each of the publications cited herein is also incorporated herein by reference] and cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and lOOμg/mL streptomycin sulfate. Pro viral DNA of T-tropic HIV-I strain HXBc2 was transfected into Jurkat cells to generate viral inoculums by the DEAE- Dextran method. HIV-I HXBc2 expresses HIV-I Eli strain nef and all other HXB2 genes. Unless stated otherwise, all chemicals were from Sigma (St. Louis, MO). EXAMPLE 3

HIV- 1 replication assay. One million Jurkat cells in 1 ml culture medium were infected with HIV-I corresponding to a 10,000 cpm RT activity. At twenty- four hr post-infection, cells were treated with plant extracts at indicated concentrations or equivalent concentrations of the DMSO solvent. Fresh extracts as well as DMSO were added every other day. Meanwhile, the culture supernatants were collected for the RT activity assay. Briefly, 1 ml of the culture supernatant was collected and cleared to remove any cells and cell debris by centrifugation at 1,000 g for 5 min, followed by filtration of the cleared supernatants through a 0.2 μm syringe filter. Virions in the supernatant were pelleted by centrifugation at 12,000 g for 1 hr and the RT activity was determined as described [Li J, et al., J Virol 2002, 76:4526-4535; Liu Y, et al., J Biol Chem 2004].

EXAMPLE 4

Cytotoxicity and syncytia formation. The cytotoxicity of the plant extracts was determined using the trypan blue exclusion method. Briefly, Jurkat cells that were exposed to plant extracts in the presence or absence of HIV-I infection for various lengths of time were stained in 0.2% trypan blue dye and then counted for viable cells under a light microscope. HIV-I -infected Jurkat cells were scored for syncytia formation from 4 random fields from each of the triplicate samples over the course of HIV- 1 infection by a light microscope.

EXAMPLE 5

Preparation and infection of HIV-I pseudotyped viruses. HIV-I viruses pseudotyped with different envelope proteins were prepared as previously described [He J, et al, Nature 1997, 385:645-649; Li J, et al, J Virol 2002, 76:8374-8382]. Briefly, 293T cells (2 xlO⁶ cells per 10-cm plate) were transfected with 20 μg of HIV-Luc plasmid and 4μg of pHXB2-env, p89.6-env, pVSV-G, or pcDNA3 by the calcium phosphate precipitation method. Cell culture supernatants were collected 48 hr after medium change, filtered, and saved as virus stocks. For infection, pseudotyped viruses corresponding to a 2,000 cpm RT activity were used to infect target cells. Following 2 hr infection, the cells were removed of remaining viruses by multiple washes with fresh medium. The cells were continued to incubate for 48 hr and then harvested for the Luc activity assay as described [He J, 1997; Li J, 2002].

EXAMPLE 6

Fractionation of crude extracts of TXE and VAD. TXE and VAD extracts obtained above were suspended in 1.5 L H₂O and partitioned successively with petroleum ether (PE) (4 x 1.5 L), chloroform (CF) (5 x 1.5 L), ethyl acetate (EA) (5 x 1.5 L), and n- butanol (BT) (5 x 1.5 L) to obtain respective subfractions. The excessive solvents were removed from these subfractions under a reduced pressure to generate ointments. The ointments were lyophilized to the powder form. These subfractions were dissolved in DMSO at a concentration of 100 mg/mL by overnight shaking on a shaker, and the undissolved materials were removed by low speed centrifugation followed by filtration through syringe filter, as described above.

EXAMPLE 7

Data analysis. All values expressed as mean ± SEM, or representative of at least three independent experiments. Comparisons among groups were made using two- tailed Student's t-test. A p value of < 0.05 was considered statistically significant (*), and p < 0.01 highly significant (**). While certain embodiments of the present invention have been described and/or exemplified above, it is contemplated that considerable variation and modification thereof are possible. Accordingly, the present invention is not limited to the particular embodiments described and/or exemplified herein.

Claims

WHAT IS CLAIMED IS:

1. An active fraction useful for treating HIV infection obtained from plant material comprising one or more plant parts of at least one plant selected from the group consisting of Euphorbiaceae, Trigonostema xyphophylloides, Dipterocarpaceae, Vatica astrotricha, Annonaceae, Artabotrys pilosus, and Annonaceae, Dasymaschalon rostratum by a process comprising the step of extracting the plant material with a first extraction agent..

2. The active fraction of claim 1 wherein the process further comprises the steps of drying the plant material and grinding the plant material, wherein the drying step precedes the extracting step.

3. The active fraction of claim 2 wherein the first extraction agent comprises a polar or a non-polar solvent, or a mixture thereof.

4. The active fraction of claim 2 wherein the first extraction agent comprises a mixture of ethanol and water.

5. The active fraction of claim 2 wherein the extraction agent comprises a mixture of from 70% to 95% ethanol and from 30% to 5% water.

6. The active fraction of claim 2 wherein the extraction is performed at a temperature of from 75° C to 85⁰C.

7. The active fraction of claim 2 in the form of a concentrate wherein the process further comprises the step of removing the first extraction agent by

evaporation.

8. A second active fraction obtained by a process comprising the step of extracting the concentrate of claim 7 with a second extraction agent.

9. The second active fraction of claim 8 wherein the second extraction agent is a polar solvent, a non-polar solvent, or a combination thereof.

10. The second active fraction of claim 8 wherein the second extraction agent is petroleum ether, methylene chloride, ethyl acetate, n-butanol, or a combination thereof.

11. The active fraction of any one of claims 1 to 10 wherein the plant is Euphorbiaceae, Trigonostema xyphophylloides.

12. The active fraction of any one of claims 1 to 10 wherein the plant is Dipterocarpaceae, Vatica astrotricha.

13. The active fraction of any one of claims 1 to 10 wherein the plant is Annonaceae, Artabotrys pilosus.

14. The active fraction of any one of claims 1 to 10 wherein the plant is Annonaceae, Dasymaschalon rostratum.

15. The active fraction of any one of claims 1 to 10 wherein the plant part is root.

16. The active fraction of any one of claims 1 to 10 wherein the plant part is stem.

17. The active fraction of any one of claims 1 to 10 wherein the plant part is leaf.

19. The process of claim 18 wherein the process further comprises the step of drying the plant material, wherein the drying step precedes the extracting step.

20. The process of claim 19 wherein the process further comprises the step of grinding the plant material.

21. The process of claim 20 wherein the first extraction agent comprises a polar or a non-polar solvent, or a mixture thereof.

22. The process of claim 20 wherein the first extraction agent comprises a mixture of ethanol and water.

23. The process of claim 20 wherein the extraction agent comprises a mixture of from 70% to 95% ethanol and from 30% to 5% water.

24. The process of claim 20 wherein the extraction is performed at a temperature of from 75° C to 85⁰C.

25. The process of claim 20 wherein the process further comprises the step of removing the first extraction agent by evaporation to yield a concentrate.

26. The process of claim 25 wherein the process further comprises the step extracting the concentrate with a second extraction agent.

27. The process of claim 26 wherein the second extraction agent is a polar solvent, a non-polar solvent, or a combination thereof.

28. The process of claim 26 wherein the second extraction agent is petroleum ether, methylene chloride, ethyl acetate, n-butanol, or a combination thereof.

29. The process of any one of the claims 18 to 28 wherein the plant material is from Euphorbiaceae, Trigonostema xyphophylloides.

30. The process of any one of the claims 18 to 28 wherein the plant material is from Dipterocarpaceae, Vatica astrotricha.

31. The process of any one of the claims 18 to 28 wherein the plant material is from Annonaceae, Artabotrys pilosus.

32. The process of any one of the claims 18 to 28 wherein the plant material is from Annonaceae, Dasymaschalon rostratum.

33. The process of any one of the claims 18 to 28 wherein the plant material comprises a part of the plant selected from the group consisting of root, stem, leaf, seeds, and flowers.

35. The composition of claim 34 wherein the process further comprises the step of drying the plant material wherein the drying step precedes the extracting step.

36. The compostion of claim 35 wherein the process further comprises the step of grinding the plant material.

37. The composition of claim 36 wherein the first extraction agent comprises a polar or a non-polar solvent, or a mixture thereof.

38. The composition of claim 36 wherein the first extraction agent comprises a mixture of ethanol and water.

39. The composition of claim 36 wherein the extraction agent comprises a mixture of from 70% to 95% ethanol and from 30% to 5% water.

40. The composition of claim 36 wherein the extraction is performed at a temperature of from 75° C to 85⁰C.

41. The composition of claim 36 wherein the process further comprises the step of removing the first extraction agent by evaporation to yield a concentrate.

42. The composition of claim 41 wherein the process further comprises the step extracting the concentrate with a second extraction agent.

43. The composition of claim 42 wherein the second extraction agent is a polar solvent, a non-polar solvent, or a combination thereof.

44. The composition of claim 42 wherein the second extraction agent is petroleum ether, methylene chloride, ethyl acetate, n-butanol, or a combination thereof.

45. The composition of any one of claims 34 to 44 wherein the plant material is from Dipterocarpaceae, Vatica astrotricha.

46. The composition of any one of claims 34 to 44 wherein the plant material is from Annonaceae, Artabotrys pilosus.

47. The composition of any one of claims 34 to 44 wherein the plant material is from Annonaceae, Dasymaschalon rostratum.

48. The composition of any one of claims 34 to 44 wherein the plant material comprises a part of the plant selected from the group consisting of roots, stems, leaves, seeds, and flowers.

49. A method for treating a patient in need of relief from HIV infection, the method comprising the step of administering to the patient a therapeutically effective amount of the active fraction of any one of claims 1 to 10 or the pharmaceutical composition of any one of claims to 34 to 48.