INVESTIGATION ON REGIONAL VARIATION IN TOTAL PHENOLIC, ALKALOID CONTENT AND IN-VITRO ANTIOXIDANT ACTIVITY OF LEUCAS ASPERA

INVESTIGATION ON REGIONAL VARIATION IN TOTAL PHENOLIC, ALKALOID CONTENT AND IN-VITRO ANTIOXIDANT ACTIVITY OF LEUCAS ASPERA

Ganga Rao B *, P. Rajeswararao, P. Prayaga Murty, E. Sambasiva Rao, P. Madhukiran, T. Mallikarjuna Rao and V. S. Praneeth D

NAIP/ICAR Sub-Project, A.U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam- 530003, Andhra Pradesh, India

ABSTRACT

In this study, we investigated and compared the total phenolic, alkaloid content and in-vitro antioxidant activity of  Leucas aspera collected from four different regions; Tirupathi (Southern zone), Lam (Krishna river region), Jagityala (Northern Telangana) and Hyderabad (Southern Telangana) of Andhra Pradesh, India. Quantitative regional variation was observed in total phenolic content, and alkaloid content in methanolic extracts of Leucas aspera from above four regions of Andhra Pradesh. Concentration dependent antioxidant activity was observed for all these extracts and also observed regional variation for scavenging of Superoxide, Hydroxyl and DPPH radicals. Among the four regions, Leucas aspera from Jagityala region contains more phenolic content (48.06±0.4µg/100 µg), Tirupathi region contains good alkaloid content (58.6±0.1µg/mg) and Hyderabad zone showed better free radical scavenging activity (IC₅₀ value for superoxide radical 156.34µg, Hydroxyl radical 122.34µg and DPPH radical 57.12µg respectively).

Antioxidant  activity

INTRODUCTION: Leucas aspera (Lamiaceae) is an annual, branched herb, erecting weed to a height of 15-60 cm with stout and hispid acutely quadrangular stem and branches. This weed is distributed throughout India from Himalayas down to Ceylon ¹. This weed is used traditionally as antipyretic and insecticide. Bruised leaves are applied locally in snake bites ^{2, 3}.

The plant contains various phytochemical constituents mainly triterpenoids, Ursolic acid and β-sitosterol, Nicotine, Sterols, glucoside, diterpenes, oleanolic acid, ursolic acid, phenolic compounds [4-(24-hydroxy-1-oxo-5-n-propyltetracosantyl)-phenol] ^4-11.

In this investigation, a detailed study was carried out on regional variation in total phenolic and alkaloid contents of the alcoholic extracts of L. aspera collected from different regions of Andhra Pradesh like Tirupathi, Lam, Hyderabad and Jagityala, and were screened for in vitro free radical scavenging activity of superoxide radical, Hydroxyl radical and DPPH radical.

MATERIAL AND METHODS:

Chemicals: All the chemicals and reagents used were of analytical grade. Folin-Ciocalteu reagent, 1, 1- diphenyl-2-picrylhydrazyl was purchased from Sigma Chemical Company, St. Louis, USA), Riboflavin from Loba Chemie Pvt. Ltd., Bombay, Deoxyribose, Bromo cresol green and Nitroblue tetrozolium were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai.

Plant Material: The plant material was collected from different climatic zones of Andhra Pradesh i.e. Tirupathi, Lam, Jagityala and Hyderabad. The Voucher specimens were deposited in the herbarium, College of Pharmaceutical Sciences, Andhra University.

Preparation of Extract: The freshly collected leaves of the plant were shade dried and powdered. The powdered material was then subjected to triple maceration with methanol: water (70:30). The extract thus obtained was concentrated under vacuum at temperature of 43^oC by using rotary evaporator (Buchi), dried completely, weighed and stored in desiccators.

Total Phenolic content: Total phenolic content was estimated as GAE (GA equivalents) as described by Singleton, Orthofer and Lamuela-Raventos, 1999 ¹². Briefly, 100µl aliquot of dissolved extract was transferred to 10ml volumetric flask containing 6.0ml ultra pure water, to which was subsequently added 500µl undiluted Folin-Ciocalteu reagent. After 1min, 1.5ml 20g/100ml sodium carbonate was added and the volume was made up to 10ml with ultra pure water. After 30 minutes incubation at 25^oC the absorbance was measured at 760nm and compared to GA calibration curve. All experiments were performed thrice; the results were averaged and reported in the form of Mean ± S.E.M (Table 1).

Total Alkaloid Content: Total alkaloid content was determined by the Fazel et al., 2008 method ¹³.The plant extract (1mg/ml) was dissolved in 2 N HCl and then filtered. The pH of phosphate buffer solution was adjusted to neutral with 0.1 N NaOH. One ml of this solution was transferred to a separating funnel and then 5 ml of Bromocresol green solution along with 5 ml of phosphate buffer were added.

The mixture was shaken and the complex formed was extracted with chloroform by vigorous shaking. The extracts were collected in a 10 ml volumetric flask and diluted to volume with chloroform. The absorbance of the complex in chloroform was measured at 470 nm. All experiments were performed thrice. The results were averaged and reported in the form of Mean ± S.E.M. (Table 1).

In-vitro Antioxidant Activity: The alcoholic   extracts of Leucas aspera from four regions were screened for free radical scavenging activity against Superoxide radical, Hydroxyl and DPPH radicals at different concentrations. The Percentage Inhibition and 50% Inhibition Concentration’s (IC₅₀) were calculated. All experiments were performed thrice and the results were averaged.

Superoxide Radical Scavenging Activity: Superoxide radical scavenging activity of the extract was measured according to McCord and Fridovich method, 1969 ¹⁴. It depends on light induced superoxide generation by riboflavin and the corresponding reduction of nitroblue tetrazolium. All the solutions were prepared in phosphate buffer (pH 7.8). The optical density was measured at 560nm.

Hydroxyl Radical Scavenging Activity: Hydroxyl radical scavenging activity was measured according to the method of Elizabeth and Rao, 1990 ¹⁵, by studying the competition between deoxyribose and test extract for hydroxyl radicals generated by Fenton’s reaction. The damage imposed on deoxyribose due to the free radicals was determined calorimetrically by measuring the thiobarbituric acid reactive substances (TBARS) at 532 nm.

DPPH Radical Scavenging Activity: DPPH radical scavenging activity was measured according to the method of Braca et al., 2003 ¹⁶. An aliquot of 3ml of 0.004% DPPH solution in ethanol and 0.1ml of plant extract at various concentrations were mixed and incubated at 37˚C for 30 min. and absorbance of the test mixture was read at 517nm.

Calculation of Percentage Inhibition: The percentage inhibition of superoxide production by the extract was calculated using the formula:

Inhibitory ratio =   (Ao-A₁)   × 100

A₀

Where, A₀ is the absorbance of control; A₁ is the absorbance with addition of plant extract/ ascorbic acid.

Calculation of 50% Inhibition Concentration: The optical density obtained with each concentration of the extract/ascorbic acid was plotted taking concentration on X-axis and percentage inhibition on Y-axis. The graph was extrapolated to find the 50% inhibition concentration of extract/ ascorbic acid.

Statistical Analysis: Values were expressed as means ± standard deviation. Analysis of variance was conducted and differences between variables were tested for significance by one-way ANOVA and linear regression analysis was used to calculate IC₅₀ values. All determinations were done at least in triplicate and all were averaged.

RESULTS AND DISCUSSION:

Total Phenolic content: The alcoholic extracts of Leucas aspera collected from different climatic regions of Andhra Pradesh i.e., Tirupathi, Lam, Hyderabad and Jagityala showed variation in Phenolic content (Tirupathi- 3.6±0.1, Lam- 44.5±0.2, Hyderabad-38.2±0.2 and Jagityala-48.06±0.4 mg/gm). Hyderabad region contains more phenolic content compared to other regions (Table 1).

Total Alkaloid Content: Regional variation in total alkaloid content also observed in four regions of leucas aspera extracts i.e Tirupathi- 58.6±0.1, Lam-24.1±0.3, Hyderabad-28.5±0.2 and Jagityala- 22.4±0.3 mg/gm respectively. Tirupathi region contains more alkaloid content compared to other regions (Table 1).

TABLE 1: REGIONAL VARIATION IN TOTAL PHENOLIC AND ALKALOID CONTENT OF LEUCAS ASPERA FROM DIFFERENT REGIONS

Superoxide Radical Scavenging Activity: Superoxide anion plays an important role in the formation of more reactive species such as hydrogen peroxide, hydroxyl radical, and singlet oxygen, which induce oxidative damage in lipids, proteins, and DNA. Therefore, studying the scavenging activity of plant extracts/compounds on superoxide radical is one of the most important ways of clarifying the mechanism of antioxidant activity. The extracts of Leucas aspera produced concentration dependent inhibition of superoxide anion i.e. IC₅₀ value of L. aspera from Tirupathi region-670.25, Lam-540.12, Hyderabad-156.34 and Jagityala- 380.24µg/0.1ml respectively (Table 2, Table 5, Fig. 1 and Fig. 4). Leucas aspera from Hyderabad region showed better inhibition of superoxide radicals.

TABLE 2: IN-VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF SUPEROXIDE RADICAL BY ALCOHOLIC EXTRACTS OF LEUCAS ASPERA AND ASCORBIC ACID

FIG. 1: IN-VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF SUPEROXIDE RADICAL BY ALCOHOLIC EXTRACTS OF LEUCAS ASPERA (LA) AND ASCORBIC ACID

Hydroxyl Radical Scavenging Activity: A single hydroxyl radical can result in formation of many molecules of lipid hydroperoxides in the cell membrane, which may severely, disrupts its function, and lead to cell death. The extracts of Leucas aspera from four regions produced concentration dependent inhibition of hydroxyl radical i.e. IC₅₀ value of L. aspera from Tirupathi region-304.25, Lam-210.26, Hyderabad-122.34 and Jagityala-252.12 µg/ml respectively (Table 3, Table 5, Fig. 2 and Fig. 4). Leucas aspera from Hyderabad region showed better inhibition of hydroxyl radicals.

TABLE 3: IN-VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF HYDROXYL RADICAL BY ALCOHOLIC   EXTRACTS OF LEUCAS ASPERA AND ASCORBIC ACID

 FIG. 2: IN-VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF HYDROXYL RADICAL BY ALCOHOLIC EXTRACTS OF LEUCAS ASPERA (LA) AND ASCORBIC ACID

DPPH Radical Scavenging Activity: All extracts from four regions showed better inhibition of DPPH radicals (IC₅₀ value of L. aspera from Tirupathi region-280.34, Lam-55.16, Hyderabad-57.12 and Jagityala-240.32 µg/ml respectively) and this is concentration dependent (Table 4, Table 5, Fig. 3 and Fig. 4). Leucas aspera from Lam, Hyderbad regions showed better inhibition of DPPH radicals. Preliminary phytochemical screening of the alcoholic   extracts of L. aspera showed the presence of alkaloids, saponins, carbohydrates, phytosterols, terpenoids and flavonoids. Natural antioxidants such as plantphenols, flavonoids and tannins possess potent antioxidant activity ¹⁷. Sterols like β-sitosterol ¹⁸, terpenoids ¹⁹, oleanolic acid and ursolic acid ²⁰ were reported to possess antioxidant activity. However, these active constituents alone or in combination may be responsible for the observed antioxidant activity.

TABLE 4: IN-VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF DPPH RADICAL BY ALCOHOLIC EXTRACTS OF LEUCAS ASPERA AND ASCORBIC ACID

TABLE 5:  IN VITRO 50% INHIBITION CONCENTRATION (IC₅₀) OF ALCOHOLIC EXTRACTS OF LEUCAS ASPERA AND ASCORBIC ACID ON FREE RADICALS SCAVENGING ACTIVITY

FIG 3: IN-VITRO CONCENTRATION DEPENDENT PERCENTAGE INHIBITION OF DPPH RADICAL BY ALCOHOLIC EXTRACTS OF LEUCAS ASPERA (LA) AND ASCORBIC ACID

FIG 4: IN VITRO 50% INHIBITION CONCENTRATIONS (IC₅₀) FOR SUPEROXIDE, HYDROXYL AND DPPH RADICAL BY ALCOHOLIC EXTRACTS OF LEUCAS ASPERA (LA) AND ASCORBIC ACID

CONCLUSION: In present study, we found clear regional variability in total phenolic and alkaloid content. Among the four regions i.e. Tirupathi, Lam, Hyderabad and Jagityala, Leucas aspera from Jagityala region contains more phenolic content; Tirupathi region contains good alkaloid content.

The results of in-vitro antioxidant activity of alcoholic extracts of L. aspera collected from different regions clearly showed regional variation in free radical scavenging activity and also produced dose dependent inhibition of free radical generation of superoxide anion, hydroxyl radical and DPPH radicals which were compared to standard antioxidant drug, ascorbic acid. Hyderabad region showed better superoxide and hydroxyl radical scavenging activity and Lam and Hyderabad region showed better inhibition of DPPH radicals. Investigation on regional variation of biological activities for these extracts is in progress.

ACKNOWLEDGMENT: The authors were thankful to World Bank funded NAIP/ICAR Sub-Project.

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