Evaluation of antioxidant and antiacne properties of terpenoidal fraction of Hemidesmus indicus (Indian sarsaparilla)
G Kumar, K Jayaveera, C Ashok, T Bharathi, S Umachigi, S Vrushabendra
antiacne activity, antioxidant activity, dpph radical, hptlc fingerprinting., nitric oxide radical, superoxide radical, terpenoidal fraction
G Kumar, K Jayaveera, C Ashok, T Bharathi, S Umachigi, S Vrushabendra. Evaluation of antioxidant and antiacne properties of terpenoidal fraction of Hemidesmus indicus (Indian sarsaparilla). The Internet Journal of Aesthetic and Antiaging Medicine. 2007 Volume 1 Number 1.
The present study was undertaken to evaluate the effect of terpenoidal fraction of the roots of
Acne vulgaris is a most common skin disorder of Pilosebaceous unit. That affects areas containing the largest oil glands, including the face, back, and trunk 16. It is generally characterized by formation of seborrhea, comedone, inflammatory lesions and presence of bacteria
In the present paper, we report our work on establishing antioxidant activity of terpenoidal fraction of
Materials and methods
Hemidesmus indicus roots were collected in month of October 2005 from tribal area of Western Ghats, Karnataka state India. Authentication of the plant material [RRI/BNG/IDNO./2005/37] was done by comparison with plant specimen located at Bangalore. Herbarium and Botanical Section of Regional Research Institute (Ayurveda), Jaynagar, Bangalore.
Preparation of extract and terpenoidal fraction
Plant material was dried at 40°C, with forced ventilation, during 3 days, before being powdered and sieved. Only material between 0.210-0.350 mm sizes was utilized. Plant material (50 g) was macerated with the solvent indicated for each species, at the ratio of 1:10 (w/v), room temperature, during 7 days. When necessary, the extracts obtained were fractionated, as described as follows. The terpenoidal fraction which contained mainly triterpenes and/or steroids was obtained by silica gel open-column preparative liquid chromatography. 23. Terpenoids was separated out and dried under vacuum (25% w/w) Vacuum-dried terpenoidal rich fraction of
Phytochemical test and TLC finger print profile of TFHI
Obtained TFHI was subjected to Liebermann Burchard test for terpenoids 24. In brief, a small quantity of the TFHI was dissolved in chloroform and treated with acetic anhydride and few drops of concentrated sulfuric acid, which gives a dark pink to red colour, indicating the presence of terpenoids, which were further separated on a thin-layer chromatography (TLC) plate and detected by treatment with anisaldehyde sulfuric acid reagent. TLC finger printing was established for TFHI using HPTLC. A stock solution of [1 mg/ml] of TFHI was prepared in chloroform. Suitable diluted stock solution was spotted on precoated plates of silica gel G60 F254 (E-Merck) using CAMAG Linomat IV automatic sample spotter and the plates were developed on solvent system of different polarities to resolve components of TFHI. The plates were dried at room temperature and scanned using CAMAG TLC scanner III at 254nm (absorbance/reflectance mode) and 366 nm (fluorescence/reflectance mode) and Rf values, spectra and peak areas of the resolved bands were recorded. Relative peak areas of each of the bands were calculated from peak areas. The TLC plates were derivatised by spraying with anisaldehyde Sulphuric acid reagent at 110° C for 5 mins for detection of terpenoids.
Free radical scavenging activity
Assay for antiradical activity
Antiradical activity was measured by a decrease in absorbance at 517 nm of a methanolic (MeOH) solution of coloured 1,1-diphenyl-2- picryl hydrazyl (DPPH) was determined by the method described by Braca et al., 25. TFHI (0.1 ml) was added to 3ml of a 0.004% MeOH solution of DPPH. Absorbance at 517nm was determined after 30 min, EC50 and the percentage inhibition activity was calculated from [(A0–A1)/A0] X 100, Where, A0 is the absorbance of the control, and A1 is the absorbance of the TFHI/ standard.
Assay for superoxide radical scavenging activity
The assay was based on the capacity of the TFHI to inhibit formazan formation by scavenging the superoxide radicals generated in riboflavin–light–NBT system 26. Each 3ml reaction mixture contained 50mM sodium phosphate buffer (pH 7.6), 20 mg riboflavin, and 12mM EDTA, and 0.1 mg NBT and 1ml TFHI solution. Reaction was started by illuminating the reaction mixture with different concentrations of TFHI (25–100 mg/ml) for 90 sec. immediately after illumination; the absorbance was measured at 590nm. The entire reaction assembly was enclosed in a box lined with aluminium foil. Identical tubes with reaction mixture were kept in the dark and served as blanks. The percentage inhibition of superoxide anion generation was calculated.
Assay of nitric oxide scavenging activity
The procedure is based on the method, where sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10mM) in phosphate buffered saline was mixed with different concentrations TFHI in methanol and incubated at room temperature for 150 min. The same reaction mixture without the methanol extract but the equivalent amount of methanol served as the control. After the incubation period, 0.5 ml of Griess reagent (1% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride was added. The absorbance of the chromophore formed was read at 546 nm. 27
Disc diffusion method
This experiment was performed by the method 20,
Determination of minimum inhibitory and bactericidal concentrations
The minimal inhibitory concentration (MIC) values were determined by broth dilution assay . The cultures were prepared at 24 h and 48 h broth cultures of
3 ml of the Nutrient yeast glucose broth (NYG) for
Preliminary chemical examination of TFHI showed an intense pink colour with the Liebermann Burchard test, indicating rich terpenoids content in the plant, which was then detected on a TLC plate by treatment with anisaldehyde sulfuric acid reagent and also characterized phytochemically by establishing its TLC finger printing profile. The TLC fingerprint profile comprises of the bands resolved, Rf values, spectral details and λ max
When scanned in UV 254 and 366 nm. Derivatisation of the TLC plates with 5 % methanolic anisaldehyde sulfuric acid reagent. The details of TLC fingerprinting profile are given in [Table 1] and the chromatogram in [Figure 1].
The TFHI exhibits different levels of antioxidant activity in all the models studied. It showed a concentration dependant antiradical activity by inhibiting DPPH radical with an EC50 value of 18.87 µg/ml (Table 2). The TFHI scavenged superoxide radicals in a dose dependent manner with EC50 value of 19.67 µg/ml, which is comparable to the activity ascorbic acid EC50 value of 17.19 µg/ml (Table 3). The TFHI also showed moderated nitric oxide scavenging activity also in a dose dependent manner with EC50 value of 43.80 µg/ml. (Table 4).
TFHI showed maximum antiacne activity the MIC values were the same (38 µg/ml) for both bacterial species and the MBC values were 38 and 46 µg/ml against
* The results indicate of average of 3 separate experiments
Free radicals have been implicated in many disease conditions, the important ones being superoxide radicals, hydroxyl radicals, peroxyl radical and singlet oxygen. Herbal drugs containing radical scavengers are gaining importance in treating such diseases.. Many plants exhibit efficient antioxidant and antiacne activities owing to their terpenoidal constituents 28, 29. In the present study, terpenoidal fraction of
DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule generally used for testing preliminary radical scavenging activity of a compound or plant extract or a fraction. In the present study THFI showed good antiradical activity by scavenging DPPH radical. It has been found that cysteine, glutathione, ascorbic acid, tocopherol, flavonoids, terpenoids, tannins, and aromatic amines reduce and decolourise DPPH by their hydrogen donating ability 29. Terpenoidal fraction of
Superoxide radical is known to be a very harmful species to cellular components as a precursor of more reactive oxygen species. TFHI was found to be an efficient scavenger of superoxide radical generated in riboflavin-NBT-system
Nitric oxide or reactive nitrogen species, formed during their reaction with oxygen or with superoxides, such as NO2, N2O4, N3O4, NO3 _ and NO2 _ are very reactive. These
compounds are responsible for altering the structural and functional behaviour of many cellular components. Incubation of solutions of sodium nitroprusside in PBS at 25 1°C for 2 h resulted in linear time-dependent nitrite production, which is reduced by the tested methanol extracts. TFHI showed a moderate nitric oxide scavenging activity when compared to DPPH radical and super oxide radical. Nitric oxide is also implicated for inflammation, cancer and other pathological conditions 30.
In conclusion, from the above investigation, using several
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