Anti-inflammatory Effect of Vitex negundo Linn. (Kyaung Pan Gyi) Leaves and Its Chemical Composition by GC-MS

Aye Aye Phyu, Ei Ei Htway, Khin May Thi, Kyaw Min Aung, Aye Aye Htun, Swe Zin Aung, Thiri Hlaing, Sandar Linn Maung Maung Thet

Myanmar Health Sciences Research Journal, 2020; 32(2):152-158

VitexnegundoLinn.(KyaungPanGyi) is a useful Myanmar medicinal plant and traditionally used with the purposes to treat rheumatism, muscular pain and muscle cramps. Plants are good sources of biologically active compounds and some of the western medicines that are used to treat inflammation are also the plant derivatives. This study was evaluated the in-vivo anti-inflammatory activity of 70% ethanolic extract of Vitexnegundo Linn. (Vn) leaves and investigated the bioactive constituents of leaf extract by Gas Chromatography-Mass Spectrometry (GC-MS). The study was done at Pharmacology Research Division, Department of Medical Research (PyinOoLwin Branch) from September 2017 to September 2018.In evaluation of the anti-inflammatory effect, the response of reduction in carrageenan-induced paw oedema in albino rat was measured by plethysmometer. A total of five groups (n=8) were tested, one negative control group (vehicle 10ml/kg), one positive control group (aspirin 300mg/kg) and three tested groups (Vn extract 250, 500 and 1000mg/kg), respectively. Among the three tested groups, leaves extract 250 mg/kg and 500 mg/kg single oral doses could not inhibit in carrageenan-induced paw oedema in rats. However, the leaves extract 1000 mg/kg inhibited the carrageenan-induced paw oedema at 2 hours (p=0.050) compared to negative control group. Acute toxicity of 70% ethanolic extract of Vn was also done by the OECD 425 guideline on albino rat and the LD50 value was found greater than 5000 mg/kg and therefore it was concluded to be safe.The bioactive phytoconstituents of the leaves extract were identified by mass spectral data of National Institute of Standard and Technology (NIST) libraries. The results from this study indicated that 70% ethanolic extract of Vn leaves is composed of medicinally useful bioactive compounds. But it did not find significant anti-inflammatory action at the tested doses.

GC-MS analysis

The GC-MS analysis of the 70% ethanolic extract of Vn leaves showed the present of many compounds.The name of compounds, its retention time and relatives area percent are shown in Table 2.

A total of 25 compounds were identified in this study. Among these compounds, the 9-octadecenoic acid which is antimicrobial and antioxidant compound23, is most prominent compound and ascorbic acids,2, 6-dihexadeconate is the second prominent compound which can give antioxidant activity.24 The other identified compounds that can give particular biological activities are also present in the Vn leaves extract with their various relatives area percent. Some of the GC-MS peaks were remained unidentified, because of lack of authentic sample and library data of corresponding compounds.

Conclusion

From this study, 70% ethanolic leaves extract of Vitexnegundo Linn. possessed most of the phytoconstituents and it had no acute toxicity up to 5000 mg/kg. Its anti-inflammatory activity was found out at 1000 mg/kg compared to that of control group (p=0.050) after 2 hours drug administration according to carrageenan-induced oedema albino rats model. The results indicated that 70% ethanolic extract of VitexnegundoLinn. leaves is composed of medicinally useful compounds. The findings of this study show that tested dose,1000 mg/kg of VitexnegundoLinn. haspotential anti-inflammatory activity
andprovide supportive information to practitioners of traditional medicine as anti-inflammatory therapy.

VitexnegundoLinn.(Family-Verbenaceae) is a useful medicinal plant and commonly known as five leaved chaste tree.9 This plant is a large woody aromatic shrub with typical five foliate pattern leaves on quadrangular branches, which give rise to bluish-purple colored flowers in branched tomentose, cymes.10 The genus Vitex contains about 270 species distributed around the world that may grow up to 6m in height. This plant is native to tropical Eastern and Southern Africa and Asia and found throughout
the greater part of India at warmer zone.11, 12

This plant is found both wild and cultivated throughout Myanmar and other South-East Asia region.This plant is used in Myanmar traditional medicine with the purposes of indigestion, dyspepsia, menstrual disorder, urinary disorder in men, rheumatism, muscular pain and muscle cramps.13

All parts of the plants are used for treatment of a wide spectrum of health disorders in traditional and folk medicine.4 Various phyto-chemical metabolites are present in the plant which make this plant useful in medical conditions.2This plant is shown to have many pharmacological activities such as anti-inflamematory activity, anti-oxidant activity, anti-bacterial activity, anti-fungal activity, skin ulcer, insecticidal activity, relieve pain of rheumatoid arthritis, hepato-protective, anti-microbial, analgesic, anti-pyretic,enlargement of the spleen, tumors and related diseases.11 Leaves of Vitexnegundo have been investigated for its anti-inflammatory activity, pain suppressing activities, anti-histamine and anti-oxidant activities.14The leaves are the most potent part of the plant for medicinal purpose.4 Plants are rich source of secondary metabolites with interesting biological activities. In general, these secondary metabolites are important in new drug development with a variety of structural classifications.15

Qualitative, quantitative and biochemical tests of secondary metabolites are the prime importance in drug evaluation.11VitexnegundoL. contains a variety of potentially bioactive compounds such as volatile oil, terpene, flavonoids, glycosides and stilbene derivatives. The major classes of compounds found in this plant are flavonoids and glycosides which possess various biological activities including anticancer, antimicrobial, antiviral, anti-inflammatory, immunomodulatory and anti-thrombotic activities.1

Gas Chromatography Mass Spectrometry (GC-MS) is the powerful analytical techniques for separation and identification of phytochemicals. Most of the anti-inflammatory compounds derived from plant sources are generally considered safe. The compounds identified by the initial qualitative analysis and GC-MS are useful in medical field. The GC-MSanalysis is the first step towards understanding the composition of medicinally active compounds in the plant. Further investigations are needed to identify the pharmacological importance and phytochemistry of the medicinal plant.17

In Myanmar, the local communities of different regions have been using Vitexnegundo Linn. to treat inflammatory diseases but it has not been approached for scientific evidence. Therefore, the present study was done to evaluate the anti-inflammatory activity of Vitexnegundo Linn. leaves on carrageenan-induced albino rat model and its chemical composition by GC-MS.

The mature leaves of Vituxnegundo Linn. (Vn) were collected from Herbal Garden, Department of Medical Research (PyinOoLwin Branch) during September and October 2017. The botanical identification of Vn was carried out by A Handbook to the Flora of Ceylon.18

Preparation of plant extract

The plant samples were washed thoroughly under running tap water to remove unwanted materials. Then, the samples were cut and allowed to dry under shaded area and ground into coarsely powder by using pounding machine. The coarse powder (50 g) was introduced into a thimble and placed in the sample holder of the Soxhlet apparatus. 70% ethanol (300 ml) was poured into flask and heated for 10 hours by using Soxhlet apparatus. The extract was concentrated under reduced pressure by using rotary evaporator and dried over digital water bath at 50ºC. The dried extract was stored in desiccators for all of the experimental procedure.

Animals

Both sexes of Wistar albino rats (180±20 g) were used for acute toxicity and anti-inflammatory activity study. The animals were produced from Laboratory Animal Service Division, DMR (POLB). The animalswere acclimatized for 5 days under laboratory conditions and fed with standard food and water ad. libitum.

Phytochemical screening

The phytochemical constituents of 70% ethanolic extract of Vn leaves were screened using the standard methods of Unani Formulation.19

Acute toxicity study

Acute toxicity test for the 70% ethanolic leaves extract of Vn was carried out by Organization for Economic Cooperation and Development (OECD) Guideline 425 on albinorat.20The starting dose was
175mg/kg and up and down dosing procedure was done until 5000 mg/kg according to main test of OECD guideline. The toxic signs were observed for each animals such as skin changes,piloerection, corneal reflex, respiratory rate, motor activity, tremor, convulsion, diarrhea, etc. The observation was done continuously during 30 minutes after dosing and special attention during 4 hours afterdosing and then daily thereafter for 14 days. Body weight changes were weekly noted. All observations were recorded for individual animal by work sheet.

Anti-inflammatory study

Acute anti-inflammatory potential of 70% ethanolic extract of Vn was investigated by using carrageenan-induced edema albino rats described by the referenced method.21 Animals were divided into five groups of six animals in each group. The groups were three tested group (250mg/kg, 500mg/kg and 1000mg/kg), and positive control group (aspirin 300mg/kg), and negative control group (vehicle 10ml/kg). Firstly, the baseline paw volumes were measured for individual animals and given calculated dose at each animal orally. One hour later, 0.1 ml carrageenan (1% in 0.9% NaCl solution) was admini- stered by subcutaneous injection into the right hind paw to induce acute inflammation. After the carrageenan injection, the paw volumes were measured by volume measuring instrument (Plethysmometer,LE 7500) at each hour after oedema induction for five hours. The percent (%) inhibition was calculated based upon the baseline paw volume. Percent inhibition was calculated by-

GC-MS analysis

Gas chromatography-Mass spectrometry GC-MS (QP2020, Shimadzu, Japan) analysis was done by using DB-5 column (30m x 0.25mm x 0.25µm film thickness) fused silica capillary column. The injection temperature was set at 280ºC and the oven temperature was initially at 45°C then programmed to 300°C at the rate of 10°C/min and held at 200°C for 5minutes. Helium was used as a carrier gas with the flow rate of1.0 ml/min. The leaves extract sample was diluted with methanol (HPLC grade) in ratio of 1:10 and 1 µl of sample was injected into the column with Auto sampler (AOC 20s) split mode in the ratio of 1:100. The mass spectrometer was operated in the Electron Ionization (EI) at 70ev. The ion source temperature was kept at 250°C with interface temperature at 280°C. The mass range was 40-800 Dalton. The compounds were identified by matching mass spectral of data with National Institute of Standard and Technology (NIST library) database.

Data analysis

The LD50 value was calculated by AOT 425 StatPgm software. The anti-inflammatory activity data was analyzed by one way analysis of variance (ANOVA) using SPSS software (version, 20). Significant value was set as p<0.05. The chemical constituents were identified by using mass spectral analysis of NIST (National Institute of Standards and Technology) library database.

ACKNOWLEDGEMENT

The authors are thankful to the Board of Directors, Department of Medical Research for permission to conduct this research, and Head of Medical Statistic Division, DMR (POLB) for data analysis.

Competing interests

The authors declare that there was no competing interests.