The antiviral effects of the neem tree
Swatantrata Kiran
Advisor: Dr. Douglas Oba
13 December 2002
Abstract
The neem tree (Azadirachta indica) has a long history of use in both Indian Ayurveda and medicine. Neem leaves were extracted into distilled water and filtered. The extract was added to herpes simplex virus type-1 while infecting Vero cells. Neem leaf extract inhibited HSV-1 replication and was not cytotoxic.
Introduction:
A number of plants are useful in medicine because of antiviral properties. In one study twenty-three different species of Nepalese plants were tested for antiviral activities against sindbis, poliovirus and herpes simplex virus. The extracts showed in vitro antiviral activity against influenza virus and herpes simplex virus (Rajbhandari et al. 2000).
The aqueous extracts from the leaves and stem of Phyllanthus orbicularis was tested for anti-viral activity in tissue culture. The extracts showed selective anti-viral activity against bovine herpes virus type 1 and type 2 (Barrio and Parra 2000). Two protein- bound polysaccharides were isolated from water soluble substances of Ganoderma lucidum and demonstrated anti-viral activity against herpes simplex virus type 1 and type 2 (Seong-kug Eo et al. 2000).
The neem tree (Azadirachta indica) has been known as the wonder tree for centuries on the Indian subcontinent (Cornborogh 2001). The history of the neem is often linked to the Indian way of life and also has a long history of use in both Indian Ayurveda and medicine and is known as the “village pharmacy” (Khan and Wassilew 1987). The neem tree is a large evergreen tree in the mahogany family. It grows naturally in India and Sri Lanka and has been successfully transplanted to other regions including West Africa, Indonesia, and Australia (Cornborogh 2001). The isolation and identification of the neem constituents began in India in 1942 (Koul et al.1989).
Neem fruits, seeds, oil, leaves, bark and roots have been used for general antiseptics antimicrobials, treatment of urinary disorders, diarrhea, fever and skin diseases (Sankaram et al.1987). The tender twigs are chewed to keep the teeth and gums healthy and the neem oil is used to cure diseases such as chronic malaria, syphilis, leprosy and other skin diseases (Dhar et al.1968). The paste of dried leaves is used to kill head lice (Dastur 1977).
The neem is also effective against certain fungi that infect the human body. Khan and Wassilew (1987) showed neem leaf extracts to be toxic to fourteen common fungi including Trichophyton, Epidermonphyton, Microsporum, Trichosporon, and Candida. Neem has also been used in India to treat several viral diseases such as smallpox, chickenpox and warts (Cornborough 2001).
Antimicrobial activity is mainly due to the chemical constituents that enable neem to protect itself from a multitude of pests. Nimbin and nimbidin found in neem have anti-viral properties and are also effective in inhibiting fungal growth on humans and animals (Cornborough 2001). Other neem extracts such as those from the kernel are anti-viral as well as inhibiting insect growth (Sankaram et al.1987).
Neem leaf extracts also reduce the viral growth cycle of vaccinia and fowl pox in chick embryo fibroblast cultures (Rai and Sethi 1972). Zeitlin et al. (1997) used various candidates including neem extracts to test for vaginal microbicides in a mouse genital herpes model. They delivered neem extracts into the vagina of the mice 20 seconds prior to delivering a highly infectious herpes simplex virus-2 inoculum. Neem was among some of the extracts, which provided significant protection.
The antiulcer effect of the aqueous extracts of the leaves of the neem tree was used in rats exposed to 2-h cold-restraint stress. The doses were administered in 10, 40, or 160mg leaf/kg body weight. The extract prevented mast cell deregulation and increased the amount of adherent gastric mucus in the stressed rats (Garg et al. 1993).
The purpose of this study was to determine the effects of leaf extracts from the neem tree (Azadirachta indica) on in vitro growth of the herpes simplex virus type 1 (HSV-1). This virus has not been previously tested with the neem leaf extract.
Materials and method:
Preparation of Leaf extract
Neem (Azadirachta indica) leaf samples were obtained and washed with distilled water. The leaves were crushed using a sterile mortar and pestle. The extract was filtered with a 0.22mfilter into a sterile vial. Extracts were used in different dilutions at 1:10, 1:50, 1:100, and 1:1000 (extract: water). Additional extracts were tested for controls. As a positive control for HSV-1 inhibition, an extract of Santalum freycinetianum was used. Likewise, Phytolacca sandwicensis, which has been shown to be ineffective against HSV-1, was used as a negative control. Neem oil (distributed by the Neem oils of Hawaii) was compared with the leaf extract. These extracts and oil were filtered and diluted 1:10, 1:50, and 1:100, as was the neem leaf extract in the second round of tests.
Cell growth
African greenback monkey fibroblasts (Vero cells) were grown in Dulbecco’s Modified Eagle’s Medium (DEM) with 10% fetal bovine serum (FBS). Cells were grown at 37C in 5% CO2 environment. Cells were plated to confluency in 6-well dishes for the antiviral assays.
Cytotoxicity of the leaf and plant extracts were tested by treating cells with 1:10, 1:50 and 1:100 dilutions of the plant extracts and incubating for three days. Cells were removed from the flask by trypsin treatment and counted for viability using 0.4% trypan blue exclusion.
Antiviral test
Dilutions of the extract were combined with HSV-1 and the virus was inoculated into triplicate wells of confluent Vero cells. The virus inoculum was at a multiplicity of infection of 0.01. The virus was allowed to attach for 1 hr at 37oC after which the virus/extract was removed and replaced with fresh DME with 2% FBS containing the same dilution of extract. The infected cells were then incubated until cytopathic effect (CPE) with visible plaques was apparent. Controls plated in triplicate, included Vero cells with similar dilutions of extract without virus and virus without extract. The number of plaques were counted and averaged for the triplicate wells.
Results and Discussion:
Vero cells treated with the extract, at the highest concentration, the 1:10 dilution did not show any cell toxicity, but an inhibition of virus infection was apparent (Table 1).
Table 1. Preliminary testing of neem extract against HSV-1
Concentration / Number of plaques (3 wells) / Average # of plaques0.1 / No plaques / No plaques
0.01 / 7832 / 2611
0.001 / 7752 / 2584
No extract / 8423 / 2808
No CPE was visible at the 1:10 dilution whereas the 1:100 and 1:1000 dilutions were clearly infected with the virus, as was the control without extract.
Fig 1. Preliminary results of infection of Vero cells in the presences of various dilution of neem extract
A quantitative analysis of the results showed a significant correlation between the concentration and the number of plaques (P= 0.003). A decrease in the concentration increased the number of plaques in the wells (fig 1).
Reducing the concentration of inoculating virus by a factor of ten, neem leaf (A. indica) extract was tested again, this time comparing several other plant extracts. Neem leaf extract was not as effective in inhibiting HSV-1 plaque formation as S. freycinetianum. However, inhibition by neem leaf extract was greater than the negative control, P. sandwicensis (fig.2)
Fig 2. Number of plaques at different concentrations from S.freycinetianu, P.sandwicensis, A. indica andno extract.
A comparison of neem leaf extract and a commercial preparation of neem oil demonstrated that the anti-viral activity is found in the leaf extract and not the oil. Leaf extract had significantly more reduction in viral plaques at 1:10 and 1:50 dilutions compared to the oil (Fig.3).
Fig 3. The number of plaques at different concentration for the neem leaf extracts and the commercial preparation of neem oil.
Although extracts of S. freycinetianum showed cell cytoxicity at the strongest concentration, neither the neem leaf extract nor the P. sandwicensis was cytotoxic (fig.4).
Fig. 4 Cell viability with various dilutions of extracts from S.freycinetianu, P.sandwicensis, A. indica andno extract.
The neem extract caused a minor decrease in viability at the highest concentration of extract, however, not enough to account for the CPE and plaque formation seen during the incubation with HSV-1.
Conclusion
The extracts from the neem leaves were effective in inhibiting the growth of the HSV-1. Virus infected cells treated with no or less than 1:10 concentration of extracts showed the most cytopathic effect. Although this study only examined the effects of a crude extract from the neem leaves, it demonstrates that certain anti-viral properties exist. Additional studies using more purified forms of the extract would give evidence to the exact nature of the anti-viral components found in the leaves, whether it is nimbin or some other component. In addition, it would be of interest to determine whether the extract is inhibiting viral binding, penetration or some other later stage in the virus replication. The neem leaf extract not being cytotoxic provides a promise that the neem leaf may prove useful in treatment of skin lesions caused by HSV-1.
Acknowledgement
I would like to thank Dr. Oba for his time, patience and help for this project. Dr. Winget for providing me with the leaves of the neem tree. Kahale Pali for S. freycinetianum extracts and Martin Rosengreen for P. sandwicensis extracts. Also Dr. Phil Bruner for the Neem oil and the BYUH Biology faculty for correcting my papers and for the helpful insights.
Works cited
Barrio, G and F Parra. 2000. Evaluation of the antiviral activity of an aqueous extract from Phyllanthus orbicularis. Journal of Ethonobotany 72: 317-322.
Cornborough, J. 2001. Neem: an ancient cure to the modern world.
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Garg, GP, SK Nigam, and CW Ogle. 1993. The gastric antiulcer effects of the leaves of the neem tree. Planta Medica 59: 215-217.
Khan, M and SW Wassilew. 1987. The effects of raw material from the Neem tree, Neem oil, and Neem extract on fungi pathogenic to humans. 3rd International Neem Conference. Nairobi, Kenya, July 10-15 pp 646-650.
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Zeitlin L, KJ Whaley, TA Hegarty, TR Moenchand and RA Cone. 1997. Tests of vaginal microbicides in the mouse genital herpes model. Contraception 56 (5): 329-35.
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