EFFECT OF ABIOTIC STRESSES ON THE MARKER CONTENT AND THE ACTIVITY OF CENTELLA ASIATICA
HTML Full TextEFFECT OF ABIOTIC STRESSES ON THE MARKER CONTENT AND THE ACTIVITY OF CENTELLA ASIATICA
Hurmat, Pargat Singh, Gulshan Bansal and Richa Shri *
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala - 147002, Punjab, India.
ABSTRACT: Centella asiatica (CA; family Apiaceae) is extensively used in traditional medicine. A regular supply of this plant is required by the herbal drug industry. Hence, the cultivation of this plant is required. The emphasis of the available cultivation practices is on biomass yield. The activity of the plant, however, depends on the amount of bioactive constituents. The production of these secondary metabolites is influenced by biotic as well as abiotic factors. This study was carried out to determine the abiotic factors that enhance the production of secondary metabolites in CA. CA plants were grown in two seasons (i.e., season 1: August to December and season 2: February to June) on different soil types (Clay loamy, Red, Black), and various stresses were applied to the plants. At the end of each season, plant yield, extract yield, asiatic acid content, antioxidant, and acetylcholinesterase inhibition activities were determined. Asiatic acid content was determined by an HPLC method. The results show the varied response of CA plants to seasons, soil, and stresses. CA plants grown in season 2, on black soil under salt stress have the highest asiatic acid content and AChE inhibition. Hence, these conditions may be recommended for incorporation in the cultivation practices of this valuable medicinal plant. This would ensure the commercial supply of plants with higher asiatic acid content and better activity.
Keywords: |
Abiotic stress, Asiatic acid, Centella asiatica, Marker content, Antioxidant activity
INTRODUCTION: Medicinal plants have an important role in the health care system and have immense potential in domestic and international market 1, 61. Approximately 70-80% of the world population uses herbal medicines for health management because of better acceptability and lesser side effects 2, 52. The demand for medicinal plants for use in herbal formulations has been increasing day- by- day; thus, a sustainable supply of medicinal plants to meet the market demand is of prime importance, which can be fulfilled by developing optimum cultivation conditions.
The therapeutic potential of herbal drugs has been attributed to the presence of various chemical constituents. It is well documented that different environmental factors particularly, abiotic factors including temperature, shade, minerals, drought, flooding, and salinity, affect plant growth, quality, and quantity of phytoconstituents 3, 4, 53, 54. Thus, manipulation of environmental factors may lead to enhanced secondary metabolite biosynthesis and thus increasing the medicinal value of plants 5, 62.
Centella asiatica L. Urban (CA) (family Apiaceae) is a perennial herbaceous, creeper, and hardy medicinal plant used in different traditional medicinal systems since ancient times 6, 55. In Ayurveda, CA (commonly known as Mandukparni or Gotu Kola) is documented as a “miracle elixir of life” 7, 63. It is described as a psychoactive medicinal plant that has been used as a brain tonic, memory enhancer, neuroprotective, anxiolytic, anti-alzheimer and anti-depressant 8, 9, 64. Scientific studies showed numerous pharmacological activities of CA, including memory enhancing and learning, neuroprotective, anxiolytic, anti-inflammatory, wound healing, cardiovascular protective and anti-cancer, thereby validating its traditional claims 7, 10, 11, 56, 63. The plant contains various phytoconstituents viz. triterpene (asiatic acid, madecassic acid, brahmic acid, isobrahmic acid, betulic acid), saponins (asiaticosides, medacassoside, madasiatic acid), flavonoids (kaempferol, castiliferol, castilicetin), glycosides (asiaticoside A, asiaticoside B, madecassoside, centelloside) 12, 13, 14, 15, 16, 65, 68. The memory enhancing and neuroprotective effects of CA have been attributed to the presence of asiatic acid 7, 13, 17, 18 ,19, 20, 65. The cultivation practices for CA are well documented, but the emphasis has been on plant growth rather than bioactive constituents and related biological activities. Considering the medicinal importance of CA, it is worthwhile to optimize the environmental factors that enhance asiatic acid production and biological activities. Thus, the present study was designed to understand the effect of abiotic factors (seasons, soil, salinity, flooding, drought, fertilizer, shade stress) on the production of marker compounds (asiatic acid) and antioxidants and anti-acetylcholinesterase activities of CA extracts.
MATERIALS AND METHODS: The field experiment was conducted at Medicinal Plant Garden of Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India. The fresh plantlets of Centella asiatica were procured from Devi Lal Herbal Park, Yamunanagar, Haryana, India, at the start of the respective season and cultivated under different conditions.
The plant was authenticated by Haryana Forest Department, Chuhadpur Klan (Yamunanagar), with Specimen number: 27/1/2016:4; 32. Fig. 1 summarizes the plan of work of this investigation.
FIG. 1: PLAN OF WORK
Estimation of Asiatic Acid Content in all Prepared Extracts using Validated HPLC Method: Content of asiatic acid, taken as a marker compound, in different extracts was determined by an HPLC PDA method 21.
Standard solution of asiatic acid (0.1 mg/ml) in methanol was prepared and separated on a NUCLEODUR C18 (250 mm × 4.6 mm) column fitted to HPLC PDA (Waters, Milford, MA, USA) system.
A mixture of acetonitrile and phosphate buffer (20 mM, pH 3.5) (55:45% v/v) was used as a mobile phase, and detection was carried out at 206 nm. The mobile phase was filtered through a 0.45-micron membrane filter and degassed. The injection volume was 20 µl, and the flow rate was maintained at 1 ml/min.
Run time for Standard and sample was 45 min. Data acquisition was done using empower 3 software compliantly. A linear calibration plot was used for the quantification of asiatic acid in all samples, as described by Nancy et al., (2015).
LC-PDA detector analyses of standard asiatic acid of all products were carried out to establish the purity of asiatic acid peaks. Readings for each extract were taken in triplicate.
Evaluation of in-vitro Biological Activities of the Extracts:
In-vitro Antioxidant Activity: The antioxidant activity of various plant extracts was determined by the DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay 22. The IC50 value of each extract was calculated by using linear regression analysis and expressed in µg/ml. All readings were taken in triplicate.
In-vitro Acetylcholinesterase (AChE) Inhibitory Activity: The AChE inhibitory effect of each test extract was evaluated using method of Ellman et al, (1961) 23. The enzyme acetylcholinestrase hydro-lyzes the substrate acetylcholine which results in production of thiocholine. The later reacts with 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB), thereby producing 2-nitrobenzoate-5-mercaptothiocholine and %-thio-2-nitrobenzoate, which can be detected at 412 nm. All the readings were taken in triplicate.
RESULTS:
Effect of Abiotic Stresses on Plant Growth: CA is a prostrate, creeper. This can easily be propagated by stem cuttings or runners or seeds 24, 25. In this study, propagation was done by plantlets that spread fast. The plants grown under different conditions were collected, shade dried and weighed separately. Table 2 summarizes the amount of plant collected per unit area expressed as g/m2.
TABLE 2: AMOUNT OF CENTELLA ASIATICA PLANTS PER UNIT AREA GROWN UNDER DIFFERENT ABIOTIC STRESSES
Season | Soil | Yield of plants collected per unit area in both seasons expressed as (g/m2) | |||||
1 | Clay loamy | Con. 28.5 | Dro. 17.8 | Fer. 42.4 | Sh. 13.45 | S.S 7.0 | F 9.6 |
Red | Con. 15.2 | Dro. 27.5 | Fer. 22.0 | Sh. 36.9 | S.S N.A | F 6.97 | |
Black | Con. 11.3 | Dro. 12.4 | Fer. 14.0 | Sh. N.A | S.S N.A | F N.A | |
2 | Clay loamy | Con. 17.8 | Dro. 10.0 | Fer. 20.0 | Sh. 16.0 | S.S N.A | F 8.4 |
Red | Con. 20.0 | Dro. 20.0 | Fer. 15.0 | Sh. 10.0 | S.S N.A | F 5.2 | |
Black | Con. 14.2 | Dro. N.A | Fer. 16.0 | Sh. 5.0 | S.S 8.0 | F 8.0 |
Con.-Control, Dro.- Drought, Fer.- Fertilizer, Sh.- Shade, S.S- Salt stress, F- Flooding; N.G - not applicable as no growth of plants had been observed
As is seen in the results, there is no uniform pattern in terms of season or soil. In the control plants, the yield was highest in the plants grown in Season 1 in Clay loamy soil. The highest yield was observed in the group treated with fertilizers and grown in Season 1, loamy clay soil.
Some reports state that sandy loam soil is better than clay 26. According to the NMPB this plant grows over moist, fertile, loose, sandy loam and clayey soil. It grows best in monsoon periods 24.
The results of our study are similar since good growth was seen in clay loam soil during season 1, i.e., during the rainy season.
Effect of Abiotic Stresses on Centella Asiatica Extract Yield in Two Seasons: Various methods such as maceration, Soxhlet extraction, microwave-assisted, etc. are reported for the preparation of extracts from CA 27, 28, 66, 67, 68. Alcoholic or aqueous extracts of CA are generally prepared since these contain higher quantities of asiaticoside and the aglycones i.e., asiatic acid and madecassic acid 29. Thus, based on literature in our study, methanol extracts of CA were prepared by Soxhlet extraction. Table 3 summarizes the yield (% w/w, dry weight basis) of methanol extract prepared from plants grown under different conditions.
TABLE 3: YIELD OF METHANOL EXTRACTS OF CENTELLA ASIATICA PLANTS GROWN UNDER DIFFERENT ABIOTIC STRESSES
Season | Soil | Yield of methanol extracts of plants grown under different abiotic stresses in both seasons expressed as % w/w, (dry weight basis) | |||||
1 | Clay loamy | Con. 9.0 | Dro. 5.5 | Fer. 4.7 | Sh. 5.8 | S.S 5.0 | F 6.7 |
Red | Con. 10.6 | Dro. 7.0 | Fer. 14.0 | Sh. 6.0 | S.S N.A | F 3.2 | |
Black | Con. 4.0 | Dro 5.5 | Fer. 14 | Sh. N.A | S.S N.A | F N.A | |
2 | Clay loamy | Con. 7.0 | Dro. 4.1 | Fer. 3.2 | Sh. 8.2 | S.S N.A | F 7.4 |
Red | Con. 13.2 | Dro. 6.0 | Fer. 11.0 | Sh. 4.8 | S.S N.A | F 4.8 | |
Black | Con. 5.0 | Dro. N.A | Fer. 16.0 | Sh. 3.5 | S.S 5.0 | F 6.4 |
Con.-Control, Dro.- Drought, Fer.- Fertilizer, Sh.- Shade, S.S- Salt stress, F- Flooding; N.A- not applicable as no growth of plants had been observed
Phytochemical Screening of Methanol Extract of C. asiatica: The results revealed the presence of carbohydrates, tannins, flavonoids, phenols, and triterpenoids in the tested extracts.
Estimation of Asiatic Acid Content in Extracts of Different Stress Effected Plants of Centella asiatica using HPLC Method: CA builds up large quantities of pentacyclic triterpenoid saponins that include asiaticoside, centelloside, madecassoside, brahmoside, brahminoside, thankuniside, scef-foleoside, centellose, asiatic, brahmic-, centellic- and madecassic acids 30, 68. Asiaticoside and madecassoside and the sapogenin –asiatic acid are generally employed as the bioactive markers for standardization of the plant 31, 69.
The extracts of different stress effected plants of Centella asiatica of both seasons were standardized for asiatic acid content by an HPLC method as 21, 70, 71. Using the standard plot of marker compound asiatic acid Fig. 2, the asiatic acid content in various extracts was determined in Table 4.
FIG. 2: STANDARD PLOT OF ASIATIC ACID EMPLOYING AN HPLC ANALYSIS
TABLE 4: ASIATIC ACID CONTENT IN EXTRACTS OF DIFFERENT STRESS EFFECTED PLANTS OF CENTELLA ASIATICA USING HPLC METHOD
Season | Soil | Asiatic acid content in methanol extracts of different stress effected plants (µg/ml) (Meann ± S.D.) | |||||
1 | Clay loamy | Con.
19.18 ± 0.78 |
Dro.
7.29 ± 0.13 |
Fer.
8.44 ± 0.42 |
Sh.
12.16 ± 0.20 |
S.S
14.11 ± 0.18 |
F 8.74 ± 0.05 |
Red | Con.
15.77 ± 0.48 |
Dro.
12.49 ± 0.18 |
Fer.
2.30 ± 0.07 |
Sh.
9.62 ± 0.21 |
S.S
NA |
F
NA |
|
Black | Con.
15.13 ± 0.19 |
Dro.
11.53 ± 0.79 |
Fer.
11.09 ± 0.53 |
Sh.
N.A |
S.S
N.A |
F
N.A |
|
2 | Clay loamy | Con.
16.06 ± 0.9* |
Dro.
8.57 ± 0.91 |
Fer.
8.32 ± 0.66 |
Sh.
18.64 ± 0.8* |
S.S
N.A* |
F
2.81 ± 0.5* |
Red | Con.
8.51 ± 0.54* |
Dro.
NG* |
Fer.
0.17 ± 0.13* |
Sh.
7.92 ± 0.83 |
S.S
N.A |
F
56.94 ± 0.11* |
|
Black | Con.
21.06 ± 0.9* |
Dro.
NA* |
Fer.
21.92 ± 0.99* |
Sh.
39.29 ± 0.96* |
S.S
66.76 ± 0.99* |
F
45.85 ± 0.50* |
The data was expressed as mean ± S.D and analyzed by student t-test of independent samples,*= p<0.05 vs.S1, n = 3, Con.-Control, Dro.- Drought, Fer.- Fertilizer, Sh.- Shade, S.S- Salt stress, F- Flooding; N.A- not applicable as no growth of plants had been observed
FIG. 3: COMPARISON OF ASIATIC ACID CONTENT OF CONTROL PLANTS GROWN IN SOIL A, B AND C IN BOTH SEASONS. The data is expressed as Meann ± S.D. (n=3) and analyzed by One way ANOVA followed by Tukey’s post hoc analysis. ap<0.05 vs. soil A season 1; bp<0.05 vs. soil A season 2; c p<0.05 vs. soil B season 1; dp<0.05 vs. soil B season 2; ep<0.05 vs. soil C season 1.
FIG. 4: ASIATIC ACID CONTENT OF PLANTS GROWN UNDER DIFFERENT STRESSES IN SEASON 2 AND SOIL C. The data is expressed as Meann ± S.D. (n=3) and analyzed by one way ANOVA followed by Tukey’s post hoc analysis. ap<0.05 vs. control.
In season 2, plants grown under different abiotic stresses showed statistically significant variation in asiatic acid content in comparison to control plants Fig. 4. The methanol extract prepared from salinity effected plants grown in soil C in season 2 contains the highest asiatic acid content.
Evaluation of Bioactivities:
In-vitro DPPH Free Radical Scavenging Activity of Extracts of Different Stress Effected Plants of Both the Seasons: The antioxidant activity of extracts of different stress effected plants of Centella asiatica of both the seasons were evaluated using in-vitro DPPH assay. The IC50 values are reported in Table 5.
The antioxidant activity of extracts prepared from different stress effected plants of both seasons is significantly different from control plants. The plants grown in Season 2 in all soil types had higher antioxidant activity than the plants grown in Season 1.
In season 1, most significant activities was observed in extracts of plants grown under controlled conditions and under salt stress in clay loamy soil (i.e. soil A). The plants grown in fertilized red soil show strong antioxidant activity in season 2. Salinity effected plants in season 1 have the highest antioxidant potential.
TABLE 5: THE IC50 VALUE IN DPPH ASSAY OF THE PLANTS OF BOTH THE SEASONS EFFECTED BY DIFFERENT STRESSES
Soil | Sample | IC50 value (µg/ml) (Meann ± S.D)
Season 1 |
IC50 value (µg/ml) (Meann ± S.D)
Season 2 |
Ascorbic acid (Standard) | 5.2 ± 0.6 | 5.2 ± 0.6 | |
A |
Control plant | 48.68 ± 0.06 | 48.63 ± 0.05 |
Drought stress | 109.00 ± 0.15 | 109.16 ± 0.15 | |
Fertilization stress | 78.63 ± 0.12 | 53.23 ± 0.15 | |
Salt stress | 42.03 ± 0.42 | N.A | |
Flooding stress | 60.75 ± 0.26 | 60.72 ± 0.23 | |
Shade stress | 78.56 ± 0.05 | 58.08 ± 0.30 | |
B |
Control plant | 51.6 ± 0.4 | 71.53 ± 0.11 |
Drought stress | 73.14 ± 0.27 | 73.11 ± 0.27 | |
Fertilization stress | 70.94 ± 0.36 | 53.36 ± 0.07 | |
Salt stress | N.A | N.A | |
Flooding stress | N.A | 69.59 ± 0.25 | |
Shade stress | 106.06 ± 0.58 | 105.4 ± 0.85 | |
C |
Control plant | 76.65 ± 0.42 | 76.65 ± 0.44 |
Drought stress | 53.46 ± 0.13 | N.A | |
Fertilization stress | 69.16 ± 0.07 | 69.13 ± 0.05 | |
Salt stress | N.A | 57.01 ± 0.31 | |
Flooding stress | N.A | 59.1 ± 0.38 | |
Shade stress | N.A | 69.09 ± 0.59 |
N.A- Not applicable as no growth of plants had been observed
TABLE 6: THE IC50 VALUE OF ACETYLCHOLINESTRASE INHIBITORY ASSAY OF THE PLANTS OF BOTH THE SEASONS
Soil | Sample | IC50 value (mg/ml) (Meann ± S.D)
Season 1 |
IC50 value (mg/ml) (Meann ± S.D)
Season 2 |
Donepezil (Standard) | 0.65 ± 0.003 | 0.65 ± 0.003 | |
A |
A Control plant | 519.7 ± 0.60 | 368.00 ± 0.76* |
A Drought plant | 431.30 ± 0.79 | 380.00 ± 0.69* | |
A Fertilized plant | 369.9 ± 0.76 | 353.00 ± 0.50* | |
A Salinity plant | 504.3 ± 0.08 * | N.A | |
A Flooding plant | 445.3 ± 0.85 | 252.00 ± 0.31* | |
A Shade plant | 454.3 ± 0.96 | 257.01 ± 0.46* | |
B |
B Control plant | 306.2 ± 0.10 | 335.00 ± 0.37 * |
B Drought plant | 431.3 ± 0.79 | 498.01 ± 0.64* | |
B Fertilized plant | 572.2 ± 0.15 | 410.01 ± 0.85 * | |
B Salinity plant | N.A | N.A | |
B Flooding plant | N.A | 175.01 ± 0.98* | |
B Shade plant | 529.1 ± 0.09 | 285.01± 0.72* | |
C | C Control plant | 361.8 ± 0.60 | 267.01 ± 0.85* |
C Drought plant | 462.6 ± 0.94* | N.A | |
C Fertilized plant | 371.7 ± 0.69 | 193.00 ± 0.91* | |
C Salinity plant | N.A | 140.01 ± 0.43* | |
C Flooding plant | N.A | 204.01 ± 0.75* | |
C Shade plant | N.A | 163.00 ± 0.50* |
The data was expressed as mean ± S.D and analyzed by t-test of independent samples,*= p<0.05 vs. S1, n = 3 Acetylcholinesterase inhibitory activity in season 2 > season 1
Acetylcholinestrase Inhibitory Activity: The acetylcholinestrase inhibitory activity of extracts of different stress effected plant of Centella asiatica of both the seasons were evaluated. The IC50 values are reported in Table 6.
The results show that the acetylcholinestrase inhibitory activity of different stress effected plants of both seasons is significantly different from control plants. Similar to acetylcholinestrase inhibitory potential than the plants grown in season 1. The different stress effected plants grown in black soil (i.e. soil C) had better acetylcholinestrase inhibitory potential than the plants of red and clay loamy soil in the season 2 Fig. 5. The salinity effected plant has the highest acetylcholinestrase inhibitory potential among all the stress effected plants in the season 2 Fig. 6.
DISCUSSION: Abiotic and biotic factors greatly effect the growth of plants and production of secondary metabolites. It is well documented that change in season, soil type (e.g. clay loamy, red, black) or variation in abiotic factors that the (i.e. light, temperature, drought, flooding, shade, salinity, fertilizer etc.) significantly influence the growth and yield of the plants 32, 72. Alteration in abiotic and biotic stresses are also responsible for change in quantity and quality of secondary metabolites. For example in Alfaalfa plants the proline content in roots is rapidly doubled under salt stress 33. Anthocyanines are reported to increase in response to salt stress 34, 57. In case of red peppers the total phenol content is increased with moderately saline level 35. This may be happen because salt stress often creates both ionic as well as osmotic stress in plants, resulting in accumulation or decrease of specific secondary metabolites in plants 36. Water deficit or drought often causes oxidative stress and was reported to show increase in the amounts of flavonoids and phenolic acids in willow leaves. Drought increased hyperforin and decreased hypericin and pseudo hypericin content in Hypericum perforatum 37. Water stress (flooding) produce changes in endogenous jasmonates in Pinus pinaster 38. Shading or low light intensity decreased the linalool and eugenol quantity and increased the methyl eugenol content in the oil of Ocimum basilicum 39. The flavonoid and phyllanthin content increased in Phyllanthus niruri due to fertilization of plant with nitrogen and magnesium 40. Compotothecin content is varied in Camptotheca acuminata along with season 41. Several studies reported that environmental factors and abiotic stress can alter plant growth, production of secondary metabolites and hence the activity of plant also effected 7, 13, 42, 43, 59. So, the study of the effect of abiotic stresses may help in increasing production of valuable secondary metabolites in medicinal plants.
CA is known as miracle elixir of life because of its vital uses from prehistoric time to the modern system of medicine. C. asiatica is cherished for its memory enhancing effect traditionally and acetylcholine esterase inhibitory activity, which is attributed to the asiatic acid present in the plant 7, 13, 17, 18, 20, 44. In the present study, the effect of abiotic stresses, seasons and different soils on the asiatic acid content and plant's antioxidant and acetylcholinesterase inhibitory activities were evaluated with a view to understand which soil to use, which season to prefer and what environmental conditions to use during cultivation to increase the production of asiatic acid in the cultivated plants and hence enhance its antioxidant and acetylcholinesterase inhibitory activity. To assess the suitability of soil for better growth of C. asiatica, the plants were grown in three different types of soils viz. clay loamy soil, red soil, black soil. It was seen that the performance with respect to growth and content due to various soil types used in the experiment differed significantly. The present data showed variation of growth in the three different soils. Plants grown in clay loamy soil have better yield with respect to biomass yield (i.e. plant/unit area). Secondary metabolite production is also influenced by different soils. For example, a study shows that the sennoside content altered among the sandy soil and black soil grown plants, the sandy soil was reported to have higher sennoside content than black soil 45.
Asiatic acid is the marker compound mainly responsible for the activity of C. asiatica 7, 13, 17, 18, 19, 20, 60. This was used as the standard in HPLC studies. The technique has been used for the determination of Asiatic acid content in plants grown under different stress parameters, in different soils and under both season on the production of the marker compound. Asiatic acid content in the control plants grown in clay loam soil was higher than the plant grows in red and black soils in seasons 1. Plants grown in black soil under the stress of salinity was found to have the better acetylcholinestrase inhibitory activity than the plants grown in clay loam soil and red soil.
Different seasons not only effect the growth but also the production of secondary metabolites in the plants 46. For example seasonal variation is responsible for alteration in essential oil yield in Ocimum basilicum 47. The growth was found to be higher in season 1 (August to December) than season 2 (February to June). The results of present investigation showed that the plants grown in season 2 have higher content of asiatic acid as compared to plants grown in season 1. The results revealed that the acetylcholinestrase inhibitory activity of all stress effected and control plants were better in season 2 than the season 1 plants. Plant growth is very much susceptible to various abiotic stresses. For instance, decrease in growth with increase in salinity 48 and water stress is well documented 49. The present study demonstrated that the plant growth was lowest in plants subjected to water stress (flooding), salinity, shade in both the seasons. C. asiatica is reported to have healthy growth in moist, swampy and rocky areas 7, thus excessive water during growth can effect the growth of plant. The present examination shows that the salt stress effected plants grown in black soil (Soil C) have highest content of asiatic acid (i.e. 39.29 µg/ml) as compared to other stress effected and control plants in season 2. Plants grown in black soil under the stress of salinity was found to have the better acetylcholinestrase inhibitory activity than the plants grown in clay loam soil and red soil. Hence, it may be concluded that it is beneficial to grow the plant C. asiatica under salt stress in black soil in season of January to June to produce higher quantities of asiatic acid in the plant.
Pharmacological investigations have reported that C. asiatica is uesd for improving cognition and relieving anxiety 7, 50, 51, 58, 60. In the present investigation the effect of abiotic factors on acetylcholinestrase inhibitory potential of plant was estimated by modifying the method proposed by Ellman et al., 1961 23, 73. The results revealed that the acetylcholinestrase inhibitory activity of all stress effected and control plants were better in season 2 than the season 1 plants. This may be due effect of seasonal variation. Plants grown in black soil under the stress of salinity was found to have the better acetylcholinestrase inhibitory activity than the plants grown in clay loam soil and red soil.
SUMMARY AND CONCLUSION: Differences in terms of plant yield, methanolic extract yield, asiatic acid content, antioxidant and acetyl-cholinesterase inhibitory activity was observed in plants grown in both seasons. HPLC studies showed that the plants of season 2 have higher asiatic acid content. In-vitro studies also showed that the plants grown in season 2 have better antioxidant and acetylcholinestrase inhibitory activity. Salinity effected plants of soil C have highest asiatic acid content, as well as acetylcholinesterase inhibitory activity among all other stress effected plants in season 2. From the results of this investigation, it may be concluded that for commercial cultivation of the plant for medicinal purposes the period from January to June is preferable. For maximum asiatic acid content and better acetylcholinesterase inhibition activity plants should be grown in black soil and given salt stress.
ACKNOWLEDGEMENT: The authors are grateful to the Department of Pharmaceutical Sciences and Drug Research, Punjabi University Patiala for providing all the facilities during the course of this study.
CONFLICTS OF INTEREST: There is no conflict of interest to declare.
REFERENCES:
- Macía MJ, García E and Vidaurre PJ: An ethnobotanical survey of medicinal plants commercialized in the markets of La Paz and El Alto, Bolivia. Journal of Ethno-pharmacology 2005; 97(2): 337-50.
- Uprety Y, Asselin H, Dhakal A and Julien N: Traditional use of medicinal plants in the boreal forest of Canada: review and perspectives. Journal of Ethnobiology and Ethnomedicine 2012; 8(1): 7-15.
- Misra N and Gupta AK: Effect of salinity and different nitrogen sources on the activity of antioxidant enzymes and indole alkaloid content in Catharanthus roseus seedlings. Journal of Plant Physi 2006; 163(1): 11-18.
- Akula R and Ravishankar GA: Influence of abiotic stress signals on secondary metabolites in plants. Plant Signaling and Behaviour 2011; 6(11): 1720-31.
- De Abreu IN and Mazzafera P: Effect of water and temperature stress on the content of active constituents of Hypericum brasiliense Choisy. Plant Physiology and Biochemistry 2005; 43(3): 241-8.
- Inamdar PK, Yeole RD, Ghogare AB and De Souza NJ: Determination of biologically active constituents in Centella asiatica. Journal of Chromatography A 1996; 742(1-2):127-30.
- Gohil KJ, Patel JA and Gajjar AK: Pharmacological review on Centella asiatica: a potential herbal cure-all. Indian Journal of Pharmaceutical Sciences 2010; 72(5): 546-53.
- Wijeweera P, Arnason JT, Koszycki D and Merali Z: Evaluation of anxiolytic properties of Gotukola–(Centella asiatica) extracts and asiaticoside in rat behavioral models. Phytomedicine 2006; 24; 13(9-10): 668-76.
- Devkota A and Jha PK: Influence of water stress on growth and yield of Centella asiatica. International Agrophysics 2011; 25: 211-14.
- Kumar MV and Gupta YK: Effect of different extracts of Centella asiatica on cognition and markers of oxidative stress in rats. J of Ethnopharmacol 2002; 79(2): 253-60.
- Rao KGM, Rao SM and Rao SG: Centella asiatica (L.) leaf extract treatment during the growth spurt period enhances hippocampal CA3 neuronal dendritic arborization in rats. Evidence-based Complementary and Alternative Medicine 2006; 3: 349-57.
- Farnsworth NR, Akerele O, Bingel AS, Soejarto DD and Guo Z: Medicinal plants in therapy. Bulletin of the World Health Organization 1985; 63(6): 965.
- Brinkhaus B, Lindner M, Schuppan D and Hahn EG: Chemical, pharmacological and clinical profile of the East Asian medical plant Centella aslatica. Phytomedicine 2000; 7(5): 427-48.
- Aziz ZA, Davey MR, Power JB, Anthony P, Smith RM and Lowe KC: Production of asiaticoside and madecassoside in Centella asiatica in-vitro and in-vivo. Biologia Plantarum 2007; 51(1): 34-42.
- Subban R, Veerakumar A, Manimaran R, Hashim KM and Balachandran I: Two new flavonoids from Centella asiatica (Linn.). Journal of Nat Medi 2008; 62(3): 369-73.
- Dora B and Khatri J: Centella asiatica: The elixir of life. International Journal of Research in Ayurveda and Pharmacy 2011; 2: 431-38.
- Zainol MK, Abd-Hamid A, Yusof S and Muse R: Antioxidative activity and total phenolic compounds of leaf, root and petiole of four accessions of Centella asiatica (L.) Urban. Food Chemistry 2003; 81(4): 575-81.
- Pohanka M, Hrabinova M, Kuca K and Simonato JP: Assessment of acetylcholinesterase activity using indo-xylacetate and comparison with the standard Ellman’s method. Int Journal of Mole Sci 2011; 12(4): 2631-40.
- Kumar GP and Khanum F: Neuroprotective potential of phytochemicals. Pharmacognosy Reviews 2012; 6(12): 81.
- Nasir MN, Abdullah J, Habsah M, Ghani RI and Rammes G: Inhibitory effect of asiatic acid on acetylcholinesterase, excitatory post synapticpotential and locomotor activity. Phytomedicine 2012; 19(3-4): 311-16.
- Bansal NY and Bansal G: HPLC-UV/FD methods for scopoletin and asiatic acid: development, validation and application in WHO recommended stability testing of herbal drug products. Biochemistry & Analytical Biochemistry 2015; 4: 1-4.
- Blois MS: Antioxidant determinations by the use of a stable free radical. Nature 1958; 181(4617): 1199.
- Ellman GL, Courtney KD, Andres Jr V and Featherstone RM: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 1961; 1: 7(2): 88-95.
- Anonymous (NMPB): Agro-techniques of selected medicinal plants. National Medicinal Plant Board, New Delhi 2014; 2: 14.
- Patel DK: Growth pattern study on Centella asiatica (L.) Urban in herbal garden. International Journal of Herbal Medicine 2015; 3(5): 09-12.
- Devkota A and Jha PK: Variation in growth of Centella asiatica along different soil composition. Botany Research International 2009; 2(1): 55-60.
- Borhan MZ, Ahmad R, Rusop M and Abdullah S: Green extraction: enhanced extraction yield of asiatic acid from Centella asiatica (L.) nanopowders. Journal of Applied Chemistry 2013.
- Azwanida NN: A review on the extraction methods use in medicinal plants, principle, strength and limitation. Journal of Medicinal and Aromatic Plants 2015; 4(196): 2167-12.
- EMA. Assessment report on Centella asiatica (L.) Urban, herba. Committee on Herbal Medicinal Products (HMPC) 2010; 30.
- James JT and Dubery IA: Pentacyclic triterpenoids from the medicinal herb, Centella asiatica (L.) Urban. Molecules 2009; 14(10): 3922-41.
- Gupta A, Verma S, Kushwaha P, Srivastava S and Aks R: Quantitative estimation of asiatic acid, asiaticoside and madecassoside in two accessions of Centella asiatica (L) Urban for morpho-chemotypic variation. Indian Journal of Pharma Education and Research 2014; 48(3): 75-79.
- Atkinson NJ and Urwin PE: The interaction of plant biotic and abiotic stresses: from genes to the field. Journal of Experimental Botany 2012; 63(10): 3523-43.
- Bakhsh A and Hussain T: Engineering crop plants against abiotic stress: current achievements and prospects. Emirates Journal of Food and Agriculture 2015: 24-39.
- Chinnusamy V, Schumaker K and Zhu JK: Molecular genetic perspectives on cross‐talk and specificity in abiotic stress signalling in plants. Journal of Experimental Botany 2004; 55(395): 225-36.
- Ramakrishna A and Gokare AR: Influence of abiotic stress signals on secondary metabolites in plants. Plant Signaling and Behaviour 2011; 6: 1720-31.
- Mahajan S and Tuteja N: Cold, salinity and drought stresses: an overview. Archives of Biochemistry and Biophysics 2005; 444(2): 139-58.
- Cramer GR, Urano K, Delrot S, Pezzotti M and Shinozaki K: Effects of abiotic stress on plants: a systems biology perspective. BMC Plant Biology 2011; 11: 163-67.
- Rejeb IB, Pastor V and Mauch-Mani B: Plant responses to simultaneous biotic and abiotic stress: molecular mechanisms. Plants 2014; 3: 458-75.
- Chang X, Alderson PG and Wright CJ: Solar irradiance level alters the growth of basil (Ocimum basilicum L.) and its content of volatile oils. Environmental and Experimental Botany 2008; 63: 216-23.
- Hanudin E, Wismarini H, Hertiani T and Sunarminto HB: Effect of shading, nitrogen and magnesium fertilizer on phyllanthin and total flavonoid yield of Phyllanthus niruri in Indonesia soil. Journal of Medicinal Plants Research 2012; 6(30): 4586-92.
- Liu Z, Carpenter SB and Constantin RJ: Camptothecin production in Camptotheca acuminata seedlings in response to shading and flooding. Canadian Journal of Botany 1997; 75(2): 368-73.
- Knight H and Knight MR: Abiotic stress signalling pathways: specificity and cross-talk. Trends in Plant Science 2001; 6(6): 262-67.
- Hawa ZE, Jaafar MHI and Fakri NFM: Impact of soil field water capacity on secondary metabolites, phenylalanine ammonia-lyase (PAL), maliondialdehyde (MDA) and photosynthetic responses of Malaysian Kacip Fatimah (Labisia pumila Benth). Molecules 2012; 17: 7305-22.
- Kumar A, Dogra S and Prakash A: Neuroprotective effect of Centella asiatica against intracerebroventricular colchicine-induced cognitive impairment and oxidative stress. International Journal of Alzheimer's Disease 2006; 8: 15-35.
- Sharma HK, Daiya KS and Chawan DD: Effect of different soil types on plant growth, leaf pigments and sennoside content in Cassia species. Pharmaceutisch Weekblad 1980; 2: 573-75.
- Mathur S, Gupta MM and Kumar S: Expression of growth and bacoside A in response to seasonal variation in Bacopa monnieri accessions. Journal of Medicinal and Aromatic Plant Sciences 2000; 22: 320-26.
- Hussain AI, Anwar F, Sherazi STH and Przybylski R: Chemical composition, an antioxidant and antimicrobial activity of basil (Ocimum basilicum) essential oils depends on seasonal variations. Food Chemistry 2008; 108: 986-95.
- Devkota A and Kumar PJ: Effect of integrated manuring on growth and yield of Centella asiatica (L.) Urban. Tropical Ecology 2013; 54: 89-95.
- Jingjing H, Chunhua LU, Xiaoming Q, Yaojian H, Zhonghui Z and Yuemao S: Effect of salinity on the growth, biological activity and secondary metabolites of some marine fungi. Acta Oceanologica Sinica 2011; 30: 118-23.
- Dhanasekaran M, Leigh AH, Angie RH, Tharakan B, Jami WP, Keith AY and Manyam BV: Centella asiatica extract selectively decreases amyloid β levels in hippocampus of Alzheimer’s disease animal model. Phytotheraphy Research 2009; 23: 14-19.
- Frederico P, Rafael CD, Dalton D, Lopes JMTP and Barbosa NR: Antioxidant and cytotoxic activities of Centella asiatica (L.) Urban. International Journal of Molecular Sciences 2009; 10: 3713-21.
- Sen S and Chakraborty R: Revival, modernization and integration of Indian traditional herbal medicine in clinical practice: Importance, challenges and future. Journal of Traditional and Complementary Medicine 2017; 7(2): 234-44.
- Ahmed AH, Darwish E and Alobaidy MG: Impact of putrescine and 24-epibrassinolide on growth, yield and chemical constituents of cotton (Gossypium barbadense L.) plant grown under drought stress conditions. Asian Journal of Plant Science 2017; 16: 9-23.
- Aftab T: A review of medicinal and aromatic plants and their secondary metabolites status under abiotic stress. Journal of Medicinal Plants 2019; 7(3): 99-106.
- Prasad A, Mathur AK and Mathur A: Advances and emerging research trends for modulation of centelloside biosynthesis in Centella asiatica (L.) Urban-A review. Industrial Crops and Products 2019; 141: 111-68.
- Razali NN, Ng CT and Fong LY: Cardiovascular Protective Effects of Centella asiatica and its triterpenes: a review. Planta Medica 2019; 20: 1203-15.
- El-Yazal MA: Presoaking treatment of propolis aqueous extract alleviates salinity stress in spinach (Spinacia oleracea L.) plants grown under calcareous saline soil conditions. International Letters of Natural Sciences 2019; 76: 23-33.
- Verma R and Gurmaita A: A review on anticarcinogenic activity of “Centella asiatica”. Endangered Species 2019; 8: 1270-80.
- Kumar I and Sharma RK: Production of secondary metabolites in plants under abiotic stress: an overview. Significances of Bioengineering and Biosciences 2018; 2: 1-5.
- Gray NE, Magana AA, Lak P, Wright KM, Quinn J, Stevens JF, Maier CS and Soumyanath A: Centella asiatica: phytochemistry and mechanisms of neuro-protection and cognitive enhancement. Phytochemistry Reviews 2018; 17(1): 161-94.
- Ravi S and Bharadvaja N: Market analysis of medicinal plants in India. Cur Pharma Bio. 2019; 20(14): 1172-80.
- Naik PM and Al-Khayri JM: Abiotic and biotic elicitors–role in secondary metabolites production through in-vitro culture of medicinal plants. Abiotic and Biotic Stress in Plants-Recent Adv and Future Perspecs 2016; 1: 247-77.
- Prakash V, Jaiswal NI and Srivastava MR: A review on medicinal properties of Centella asiatica. Asian Journal of Pharmaceutical and Clinical Res 2017; 10(10): 69-74.
- Roy A and Bharadvaja N: Centella asiatica: a pharma-ceutically important medicinal plant. Current Trends in Biomedical Engineering & Biosciences 2017; 5(3): 1-5.
- Lokanathan Y, Omar N, Puzi NN, Saim A and Idrus RH: Recent updates in neuroprotective and neuroregenerative potential of Centella asiatica. The Malaysian Journal of Medical Sciences: MJMS 2016; 23(1): 4-14.
- Jayaprakash SB and Nagarajan N: Studies on the bioactive compounds and antimicrobial activities of medicinal plant Centella asiatica (Linn). Journal of Medicinal Plants Studies. 2016; 4(5): 181-85.
- Idris FN and Nadzir MM: Antimicrobial activity of Centella asiatica on Aspergillus niger and Bacillus subtilis. Chem Engineering Trans 2017; 56: 1381-86.
- Pal RS and Pal Y: Pharmacognostic review and phyto-chemical screening of Centella asiatica Linn. Journal of Medicinal Plants Study 2016; 4(4): 132-35.
- Khemawoot P, Hengjumrut P, Anukunwithaya T, Chang LC, Wongwiwatthananukit S and Tantisira MH: Comparison of the pharmacokinetic profiles of a standardized extract of Centella asiatica and a mixture of madecassoside and asiaticoside in rats. Planta Medica International Open 2018; 5(02): 39-47.
- Rafi M, Handayani F, Darusman LK, Rohaeti E, Wahyu Y, Honda K and Putri SP: A combination of simultaneous quantification of four triterpenes and fingerprint analysis using HPLC for rapid identification of Centella asiatica from its related plants and classification based on cultivation ages. IndusCrops and Prod 2018; 122: 93-97.
- Kaur I, Suthar N, Kaur J, Bansal Y and Bansal G: Accelerated stability studies on dried extracts of Centella asiatica through chemical, HPLC, HPTLC, and biological activity analyses. Journal of Evidence Based Comple and Alternative Medicine 2016; 21(4): 127-37.
- Acosta-Motos JR, Ortuño MF, Bernal-Vicente A, Diaz-Vivancos P, Sanchez-Blanco MJ and Hernandez JA: Plant responses to salt stress: adaptive mechanisms. Agronomy 2017; 7(1): 18-23.
- Arora R, Kumar R, Agarwal A, Reeta KH and Gupta YK: Comparison of three different extracts of Centella asiatica for anti-amnesic, antioxidant and anticholinergic activities: in-vitro and in-vivo study. Biomedicine and Pharmaco-therapy 2018; 105: 1344-52.
How to cite this article:
Hurmat, Singh P, Bansal G and Shri R: Effect of abiotic stresses on the marker content and the activity of Centella asiatica. Int J Pharm Sci & Res 2020; 11(9): 4612-21. doi: 10.13040/IJPSR.0975-8232.11(9).4612-21.
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Article Information
60
4612-4621
904
638
English
IJPSR
Hurmat, P. Singh, G. Bansal and R. Shri *
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, India.
rshri587@hotmail.com
03 October 2019
20 February 2020
26 February 2020
10.13040/IJPSR.0975-8232.11(9).4612-21
01 September 2020