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73
Phytochemical Screening and Antibacterial Activity of Aloe vera Collected from Different Climatic Regions of India *
Sandeep Kumar, Lalita Budhwar, Amita Yadav, Manila Yadav and Jaya Parkash Yadav Department of Genetics, M. D. University, Rohtak-124001, Haryana, India
Abstract: In this study Aloe vera accessions were collected from 12 states covering different climatic zones of India. The aqueous crude extracts were investigated for qualitative phytochemical diversity analysis, quantitative estimation of total phenol, alkaloid and flavonoid content and their antibacterial potential against ATCC (American Type Culture Collection) bacterial reference strains. Pearsoncoefficient correlation was used to correlate the quantitative estimation between tested bio-active compounds. Antibacterial potential was evaluated by agar well diffusion method against 9 bacterial reference ATCC strains. MIC (Minimum Inhibitory Concentration) values were calculated by J.P. Yadav microbroth dilution method. Different Aloe vera accessions showed the presence of alkaloids, glycosides, reducing sugar, phenolic compounds, flavonoids and tannins. Significant correlation was observed between alkaloids and phenols, and flavonoids and alkaloid contents. The correlation analysis also revealed the significant negative correlation between temperature and phenol (-0.803), and temperature and alkaloids (-0.779). Antibacterial activities measured in term of zone of inhibition against tested bacterial strains varied in different accessions of Aloe vera. Aqueous leaf extract of Aloe vera showed good antibacterial potential against both type of bacterial strains, gram positive S. aureus as well as gram negative E. coli and S. typhi. Highland and semi-arid zone accessions showed maximum phytochemical and antibacterial potential as compared to other Aloe vera accessions. Minimum MIC value of 1.2 mg/ml was shown by Highland samples against most of the reference strains. The results also concluded that phytochemical composition of Aloe vera is influenced by various environmental factors especially temperature and it is further correlated with antimicrobial potential of the plant.
Keywords: Aloe vera, Climatic zones, Phytochemical screening, Antibacterial activity, MIC. 1. INTRODUCTION Infectious diseases treatment is a worldwide challenge due to increased resistance of microbes against broadly used antibiotics and antiviral therapies [1]. Though enormous progress has been made in the field of healthcare, still infectious diseases are the leading cause of death in developing countries [2]. There is a great need to explore for the new effective and significant natural sources of antibiotics. Natural agents do not build up antibiotic resistance, which is the major problem with the long term use of synthetic antibiotics [3]. Medicinal plants are the richest source of drugs in almost all traditional and modern medicine systems due to very less chances of adverse effects to their uses as compared to synthetic drugs [4]. Plants have been a valuable source of natural products for maintaining human health since centuries. Pharmaceuticals, nutraceuticals, food supplements and even chemical industries are strongly depending on medicinal plants [5]. Phyto-constituents present in the medicinal plants are mainly responsible for their therapeutic and medicinal importance [6]. The organic compound of plant origin which generally modulates human physiology includes phenolics, alkaloids, flavonoids, *Address correspondence to this author at the Department of Genetics, M.D. University, Rohtak-124001, Haryana, India; Tel: +91- 9416474640; E-mail:
[email protected] 2210-3155/16 $58.00+.00
tannins, terpenoids and steroids, etc. [7]. Almost 500 species, out of 1500 medicinal plants identified in Indian system are used for the purpose of drug preparations [8]. Aloe vera (L.) Burm. f. (Synonym A. barbadensis Miller) is a succulent perennial plant with green, tapering, spiny, marginated and dagger- shaped fleshy leaves filled with a clear viscous gel [9]. Aloe vera is the most commercialized Aloe species belonging to the Asphodelaceae (Liliaceae) family. It is a cactus-like plant that grows readily in hot and dry climates [10]. Dry regions of Africa, Asia, and Southern Europe, especially Mediterranean regions are the origins of these plants. Aloe vera juice has been used traditionally for its purgative effects and fresh leaf gel used in different formulations and cosmetics preparations [11]. Aloe vera contains over 75 nutrient and 200 active compounds, including vitamins, enzymes, minerals, sugar, lignin, anthraquinones, saponins, salicylic acid and amino acids, which are responsible for its medicinal properties [12]. Its secondary metabolites have multiple properties such as anti-inflammatory, antibacterial, antioxidant, immune boosting, anticancer, antiageing, sunburn relief and antidiabetic potentials [13-15]. Anthraquinones [16-18], dihydroxy- anthraquinones [19] and saponins [20] phytoconstituents of Aloe vera proposed to have direct antimicrobial activity. Aloe vera has been used since early times for the topical treatment of various skin conditions such as cuts, burns and eczema [21]. © 2016 Bentham Science Publishers
74 The Natural Products Journal, 2016, Vol. 6, No. 1
In the present study, Aloe vera plant samples were collected from 12 states covering different climatic zones of India. Aqueous crude leaf extracts of these samples were subjected to phytochemical screening; both qualitative and quantitative. Extracts were also screened for antibacterial activity against ATCC (American Type Culture Collection) bacterial strains. The purpose of the present study is to establish the correlation between phytochemical constituents of the plant with different climatic conditions and antibacterial potential of aqueous extracts prepared from leaves. 2. MATERIALS AND METHODS 2.1. Sample Collection Samples were collected from 12 sites covering 6 agroclimatic zones of India. Each zone had 2 sites. Plant collection sites are shown in Fig. 1. The average temperature and average rainfall of these sites are given in Table 1. Samples were collected in the months of Jan-Feb 2013. Identification and authentication of plant was done by comparing the herbarium specimens number MDU - 6803 available in the Department of Genetics, M. D. University, Rohtak, India. Healthy leaves of Aloe vera were collected
Kumar et al.
from individual plants at each location. Tissues were placed in sterile plastic bags. All samples were brought to the laboratory in an ice box and processed further. 2.2. Chemicals and Reagents Ethyl alcohol (absolute), methanol, chloroform, ammonia, aluminium chloride, DMSO (Dimethyl sulfoxide) hydrochloric acid (38%), sulphuric acid (95%), ferric chloride, sodium hydroxide pellets (97%), sodium chloride, sodium carbonate, sodium nitrite, phosphate buffer, lead acetate (95%), potassium acetate and gelatin (Type B, 2%) were purchased from Merck, Germany. Folin-Ciocalteu reagent, Gallic acid (97%), atropine (99%) and quercetin (95%) standards were purchased from Sigma-Aldrich Co. (St. Louis, USA). All the chemicals used were of analytical grade. 2.3. Preparation of Extracts Leaves were washed with running tap water and dried in shade. Dried leaves were crushed using a willey mill. Aqueous extracts of different accessions were prepared by cold percolation method. To 100 grams of dried leaf mass 1 L water was added in a flask (1:10). It was incubated in an incubator shaker at 28 °C for 48-72 h at 180 rpm. Extract
Fig. (1). Map showing Aloe vera accessions collection sites from different places of India.
Phytochemical Screening and Antibacterial Activity of Aloe vera Collected
Table 1.
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Plant collection sites with average temperature, rainfall and extracts yield of different Aloe vera samples.
Sr. No.
Agro-Climatic Zones
Accessions
Collection Sites
Temp. Range (oC).
Average Rainfall (mm).
Aqueous Extracts Yield(g/100g)
1
Highland
Jammu and Kashmir [J&K]
Jammu
07- 20
1,011
3.7
Himachal Pradesh [HP]
Palampur
10- 17
1,251
3.5
Haryana
Rohtak
20- 34
617
3.9
Punjab
Sangrur
15- 35
649
4.1
Rajasthan
Jaisalmer
22- 35
209.5
3.5
Gujarat
Gandhinagar
22- 33
1,107
3.8
Uttar Pradesh [UP]
Pratapgarh
19- 32
904
3.9
Madhya Pradesh [MP]
Bhopal
19- 32
1,146
3.0
West Bengal [WB]
Kolkata
22- 32
1,582
3.4
Telangana
Hyderabad
23- 30
812.5
3.7
Goa
Vasco
23- 32
3,055
3.6
Kerala
Kochi
24- 32
3,005
3.3
2 3
Semi-arid
4 5
Arid
6 7
Humid Subtropical
8 9
Tropical wet & dry
10 11
Tropical wet
12
was filtered through Whatman filter paper No. 1. Filtrate was concentrated with help of rotary vacuum evaporator at 40 °C. Dried mass was weighed. Table 1 shows the yield of extracts from different accessions. 2.4. Phytochemical Analysis 2.4.1. PhytoChemical Screening of Aloe vera Extracts Nine categories of phytochemical constituents of Aloe vera can be classified as, Anthraquinones, Inorganic Compounds, essential and non essential amino acids, fatty acids, alkaloids, carbohydrates, enzymes, and vitamins along with other miscellaneous compounds [22]. Aloe vera aqueous extracts were screened for the presence or absence of various phytochemicals according to standard protocols [23, 24]. 2.4.2. Determination of Total Phenolic Content (TPC) The total phenolic content of the obtained extracts was analyzed spectrometrically by Folin-Ciocalteu method [25]. Final volume of 5 ml was made by adding 500 µl of FolinCiocalteau reagent, 1.5 ml of 20% Na2CO3 and 2 ml of distilled water to 1 ml (1mg/ml) of each extract. The mixture was incubated at room temperature for 30-40 min. Absorbance of the developed colour was recorded at 765 nm using UVVIS spectrophotometer. Gallic acid was used as standard for TPC estimation. Calibration curve was constituted using various concentrations of gallic acid ranging from 20 to 100 µg/ml. Total phenolic value was obtained from the regression equation: y = 0.056x, R2 = 0.9967. 2.4.3. Determination of Total Alkaloid Content (TAC) The 1 mg aqueous plant extract of Aloe vera was dissolved in dimethyl sulphoxide (DMSO). After that 1ml of 2 N HCl was added and filtered. After filtration the solution
was transferred to a separating funnel, and then 5 ml of each bromocresol green solution and phosphate buffer were added to it. The mixture was shaken with chloroform (1, 2, 3 and 4 ml) by vigorous shaking in a volumetric flask and diluted to the volume with chloroform. The absorbance for test and standard solutions were determined against the reagent blank at 470 nm with an UV/Visible spectrophotometer [26, 27]. Atropine was used as standard for TAC estimation. The alkaloid contents were examined in plant extracts and expressed in terms of atropine equivalent as mg of AE/g of extract (standard curve equation: y = 0.006x, R2 = 0.996). 2.4.4. Determination of Total Flavonoid Content (TFC) Aluminum chloride method, as reported by Kale et al., was used for the estimation of total flavonoid content [28]. 0.5 ml of aqueous Aloe vera extract was dispensed into a test tube, followed by 1.5 ml of methanol, 0.1 ml of aluminum chloride (10%), 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. The reaction mixture was mixed and allowed to stand for half an hour at room temperature, before absorbance was read at 514 nm with an UV/Visible spectrophotometer. Quercetin was used as standard for TFC estimation. TFC was expressed as quercetin equivalent (QE) in mg/g material (standard curve equation: y = 0.0148x, R2 =0.975). 2.5. Antibacterial Activity 2.5.1. Reference Strains Pathogenic reference ATCC strains (maintained in our laboratory) were used for checking antibacterial activity. Nine bacterial strains were used for the study. Out of 9 bacterial strains, 7 were gram negative viz. Shigella flexneri ATCC 12022, Proteus mirabilis ATCC 43071, Salmonella typhi ATCC 13311, Serratia marcescens ATCC 27137,
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Kumar et al.
Klebsiella pneumonia ATCC 700603, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and 2 were gram positive viz. Enterococcus faecalis ATCC 29212 and Staphylococus aureus ATCC 259323.
mg tablet in 40 ml of sterile distilled water) was added in each well. A volume of 100 µl of test materials was (stock concentration 10 mg/ml of extracts) added into the first row of the plate. Serial dilutions were performed such that each well had total 100 µl of the test material in serially descending concentrations. Finally 10 µl of bacterial suspension concentration of 5 x 106 CFU/ml was added to each well. Plate had a column with gentamicin as positive control. The plates were prepared in triplicate and placed in an incubator at 37 °C for 18 to 24 h. Any colour change from purple to pink indicates growth of microbes. The highest dilution at which no color change occurred was taken as the MIC value of extract and was expressed in mg/ml.
2.5.2. In Vitro Antibacterial Assay Antibacterial activity was checked by agar well diffusion method. 100 mg of the aqueous extract was dissolved in 1 ml of DMSO and used as test sample. Overnight grown fresh culture was used for inoculum preparation. Inoculums were prepared in peptone water and incubated for 2 hours. Turbidity was adjusted equivalent to 0.5 McFarland units (approximately 108 CFU/ml). Inoculums were spread over fresh nutrient agar plates with a sterile glass spreader. Sterile cutter was used to cut 4 wells of 6 mm diameter. 10, 20, 30 and 40 µl of this prepared test sample was added to all bacterial strains used. Control disc was placed in centre as 10µg gentamicin discs were used as positive control for antibacterial activity. DMSO acted as negative control. Plates were incubated for 24 hours at 37 oC. Clear zone of inhibition around each well was measured with the help of standard ruler HiAntibiotic ZoneScaleTM-C supplied by HiMedia Laboratories Pvt. Ltd. India. Each experiment was done in triplicates.
2.6. Statistical Analysis All values are expressed as Mean ± standard deviation (SD) of three separate experiments using the computer programme MS Excel. Linear correlation between total phenolic content, total alkaloid content and total flavonoid Content were calculated with the help of MS excel programme and SPSS 16.0 version. 3. RESULTS 3.1. Phytochemical Screening
2.5.3. Minimum Inhibitory Concentration [MIC]
Alkaloids, glycosides, reducing sugar, phenolic compounds, steroids and terpenoids, flavonoids, tannins and saponin glycoside were tested with their appropriate protocols and reagents. Aloe vera aqueous extracts showed presence of mostly tested phytochemicals; though amount varied in different accessions. The comparative presence and absence of phytochemicals in different crude extracts have been depicted in Table 2.
It is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. The MIC values of extracts were determined based on a micro broth dilution method in 96 multi-well micro titer plates developed by Sarkar et al. with slight modifications [29]. 50 µl of nutrient broth and 50 µl of normal saline were added to each well of plate. 10 µl of resazurin indicator solution (prepared by dissolving a 270 Table 2.
Qualitative analyses of the phytochemical components of Aloe vera extracts (+++ = High, ++ = Medium, + = Low, --Absent).
Sr. No
PhytoChemicals
Test Reagent
Observation
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
1.
Alkaloids
Mayer’s reagent
White ppt
+++
+++
+++
+++
++
++
++
++
++
++
++
++
2.
Glycosides
10% Lead acetate solution
White ppt
++
++
++
+++
++
+
++
++
+
++
++
++
3.
Reducing sugar
Benedict’s solution
Reddish brown ppt
++
++
+
++
+
+
++
+
+
+
++
+
4.
Phenolic compounds
Ferric chloride solution
Green colour
+++
+++
++
+++
++
++
+
++
+
++
+
+
5.
Steroids, Terpenoids
Acetic anhydride and conc: H2SO4
Pink colour
---
---
---
---
---
---
---
---
---
---
---
---
6.
Flavonoids
Benzene FeCl3
Yellow ppt
++
++
++
++
++
+
++
++
+
+
++
++
7.
Tannins
FeCl3 and 10% Lead acetate
White ppt
++
++
++
++
+
+
++
+
++
+
+
+
8.
Saponin glycosides
Distilled water
Frothing take place
---
---
---
---
---
---
---
---
---
---
---
---
Phytochemical Screening and Antibacterial Activity of Aloe vera Collected
3.2. Determination of Total Phenolic Content, Total Alkaloid Content and Total Flavonoid Content All samples showed the presence of total phenolic content, total alkaloid content and total flavonoid contents in crude aqueous extracts of Aloe vera. Table 3 enlists the different values obtained for all 12 accessions. TPC values ranged from 41.9 to 53.2 mg GAE per g of dry weight for different samples. Total alkaloid content was in range from 90.5 to 111.7 mg AE per g of dry weight. Total flavonoid Table 3.
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77
content ranged from 26.4 to 37.5 mg QE per g of dry weight in different Aloe vera samples. Samples from highland (J&K and HP) and semi- arid (Haryana and Punjab) zones showed highest values for all the tested constituents while humid subtropical (UP and MP) and Tropical wet and dry (WB and Telangana) showed least values comparatively. Correlation between total phenolic content, total alkaloid content and total flavonoid content of Aloe vera extracts along with average temperature of their collection sites have been shown in Fig. 2. Alkaloids and phenols showed a significant
Quantification of phenols, alkaloids and flavonoids in Aloe vera extracts collected from different climatic zones of India. Accession Name
Total Phenolic Content (mg of GAE/g of Extract)
Total Alkaloid Content (mg of AE/g of Extract)
Total Flavonoid Content (mg of QE/g of Extract)
Jammu and Kashmir
53.2±0.15
108.4±0.07
30.5±0.41
Himachal Pradesh
52.3±0.52
111.7±0.34
31.2±0.33
Haryana
46.5±0.53
103.2±0.44
34.7±0.47
Punjab
51.3±0.03
107.0±0.67
37.5±0.23
Rajasthan
46.9±0.21
95.3±0.10
29.5±0.41
Gujarat
44.4±0.14
97.3±0.42
29.2±0.92
Uttar Pradesh
45.2±0.55
92.6±0.71
26.4±0.80
Madhya Pradesh
43.7±0.28
94.2±0.55
28.7±0.36
West Bangal
41.9±0.76
90.5±0.39
29.5±0.25
Telangana
45.5±0.35
92.2±0.11
27.3±0.72
Goa
43.5±0.05
96.0±0.84
29.9±0.02
Kerala
42.7±0.39
95.3±0.22
27.4±0.67
Fig. (2). Correlation between different bio- active constituents of Aloe vera accessions. (a) Between phenolic and alkaloid content. (b) Between phenolic and flavonoid content. (c) Between alkaloid and flavonoid content. (d) Between phenolic, alkaloid, flavonoid content and average temperature of collection.
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Kumar et al.
positive correlation (0.897). Alkaloids also exhibited positive correlation with flavonoids (0.672). However, correlation between phenols and flavonoids was not much significant. Temperature showed significant negative correlation with respect to phenols and alkaloids. Table 4 showed correlation matrices for the observed effect of temperature on quantity of different phytoconstituents.
exhibited varying degree of inhibitory effect against the selected bacterial pathogens. Each of the extract tested in the present study displayed antibacterial activity against all the bacterial strains tested; however differences were observed between activities of the extracts. Maximum zone of inhibition was displayed by highest concentration of leaf extracts of A. vera. Activity ranged from 11 to 21 mm zone of inhibition. Inhibition zones for some of the A. vera accessions against different reference strains have been given in Fig. 3. Different accessions showed variation in zone of inhibition against the same bacterial strains. Maximum activity was shown against E. coli by Punjab accession (21
3.3. Antibacterial Activity In vitro antibacterial activity of the aqueous leaf extracts of A. vera was quantitatively evaluated on the basis of zone of inhibition. All A. vera extracts in the present investigation Table 4.
Correlation matrices between different bio- active constituents of Aloe vera accessions with average temperature of their collection sites. Phenol
Alkaloid
Flavonoid
Phenol
1
Alkaloid
.897**
1
Flavonoid
.537
.672*
1
Temp.
-.803**
-.779**
-.176
Temp.
1
**Correlation is significant at 0.01 level (2-tailed). *Correlation is significant at 0.05 level (2-tailed).
Fig. (3). Plate showing zone of inhibition against different reference strains using aqueous crude extracts (100 mg/ ml) of Aloe vera. (a) Telangana accession against Pseudomonas aeruginosa. (b) UP accession against Serratia marcescens. (c) Haryana accession against Proteus mirabilis. (d) Gujarat accession against Escherichia coli.
Phytochemical Screening and Antibacterial Activity of Aloe vera Collected
Table 5.
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79
Antibacterial activity of aqueous Aloe vera extracts (mm ± SD).
Accessions
SF
EF
SA
PM
ST
SM
KP
EC
PA
J&K
18±0.7
17±0.5
16±0.1
17±1.0
18±0.5
15±0.2
16±0.5
17±0.1
18±0.6
HP
17±0.3
18±0.5
15±0.5
18±0.6
17±0.5
14±0.8
17±0.4
19±0.5
17±0.6
Haryana
17±0.5
17±0.7
16±0.3
20±0.5
18±0.7
14±0.3
18±0.3
20±0.6
20±0.5
Punjab
18±0.2
15±0.8
15±0.7
18±0.5
20±0.4
16±0.3
18±0.6
21±0.5
17±0.2
Rajasthan
16±0.5
17±0.2
15±0.9
18±0.3
17±0.9
16±0.5
17±0.2
13±0.9
18±0.5
Gujarat
14±0.3
15±0.5
13±0.2
14±0.7
14±0.4
13±0.4
14±0.5
15±1.0
14±0.7
UP
17±0.2
15±0.5
20±0.4
14±0.3
17±0.5
14±0.7
13±0.5
18±0.4
14±0.3
MP
16±0.1
14±0.2
16±0.3
13±0.5
14±0.3
14±0.5
15±0.7
17±0.7
14±0.9
WB
15±0.4
13±0.9
16±0.6
15±0.2
16±0.7
11±0.5
15±0.3
17±0.2
14±0.2
Telangana
14±0.5
13±0.4
20±0.1
14±0.6
15±0.4
16±0.1
15±0.2
20±0.3
16±0.5
Goa
15±0.3
17±0.2
15±0.5
16±0.5
13±0.5
14±0.7
16±0.7
18±0.5
15±0.9
Kerala
15±0.1
12±0.5
18±0.6
13±0.1
20±0.2
13±0.2
11±0.4
17±0.5
15±0.5
Control
23±0.4
21±0.7
24±0.4
23±1.0
24±0.7
21±0.6
20±0.5
23±0.4
21±0.5
Bacterial strains = EC- Escherichia coli, PM- Proteus mirabilis, KP- Klebsiella pneumoniae, PA- Pseudomonas aeruginosa, SF- Shigella flexneri, SM- Serratia marcescens, ST- Salmonella typhi, EF- Enterococcus faecalis and SA- Staphylococus aureus. Control for bacterial strains- Gentamicin (10µg).
Table 6.
Minimum Inhibitory Concentration (MIC in mg/ml) of Aloe vera extracts against different micro- organisms.
Accessions
SF
EF
SA
PM
ST
SM
KP
EC
PA
J&K
1.2
1.2
2.5
2.5
1.2
2.5
2.5
2.5
1.2
HP
2.5
1.2
2.5
2.5
1.2
2.5
2.5
1.2
2.5
Haryana
1.2
1.2
2.5
1.2
2.5
2.5
1.2
1.2
1.2
Punjab
1.2
2.5
2.5
1.2
2.5
2.5
1.2
1.2
2.5
Rajasthan
2.5
1.2
2.5
1.2
2.5
2.5
2.5
5.0
2.5
Gujarat
5.0
2.5
5.0
2.5
2.5
5.0
2.5
2.5
5.0
UP
2.5
2.5
2.5
2.5
1.2
2.5
2.5
2.5
2.5
MP
2.5
5.0
2.5
5.0
5.0
5.0
2.5
2.5
5.0
WB
2.5
5.0
2.5
2.5
2.5
5.0
2.5
2.5
2.5
AP
5.0
2.5
2.5
2.5
2.5
2.5
2.5
1.2
2.5
Goa
2.5
2.5
2.5
5.0
2.5
2.5
5.0
1.2
2.5
Kerala
2.5
5.0
2.5
5.0
1.2
5.0
5.0
2.5
2.5
Bacterial strains = EC- Escherichia coli, PM- Proteus mirabilis, KP- Klebsiella pneumoniae, PA- Pseudomonas aeruginosa, SF- Shigella flexneri, SM- Serratia marcescens, ST- Salmonella typhi, EF- Enterococcus faecalis and SA- Staphylococus aureus. Control for bacterial strain- Gentamicin (10µg).
mm) while minimum activity was against K. pneumonia by Kerala accession (11 mm). Inhibition zones of all the extracts have been shown in Table 5. MIC values for all extracts ranged between 1.2 to 5.0 mg/ml. J&K, HP, Punjab and Haryana accessions showed MIC values between 1.2 to 2.5 mg/ml against all tested bacterial strains. Gujarat, MP and WB accessions showed MIC in between 2.5 to 5.0
mg/ml and rest of the accessions showed wide range of 1.2 to 5.0 mg/ml MIC values against different pathogens. MIC values of all accessions are shown in Table 6. 4. DISCUSSION India is one of the richest countries in the world in regard to genetic resources of medicinal plants. It exhibits a wide
80 The Natural Products Journal, 2016, Vol. 6, No. 1
range in geography and climate along with diverse temperature and rainfall conditions, which has a bearing on its vegetation and floristic composition [30]. Phytochemical compositions of plants are greatly influenced by strong environmental and climatic fluctuations according to different seasonal and topographical changes [31]. Previous findings also suggested that different agro-climatic conditions are conducive for introducing and domesticating of different plant varieties [32]. In the present investigation, Aloe vera was collected from different agro-climatic zones of India. Aqueous leaf extracts of Aloe vera were prepared to evaluate the phytochemical diversity along with comparative antibacterial potential of the plant against different bacterial reference strains. Most of biologically active compounds of Aloe vera are present in leaves [33]. Aloe vera contains many active components some of which exert antimicrobial activities either independently or synergistically [34, 35]. Solvent type and extract preparation methods affect phytochemical concentration and antimicrobial activity of plants [36, 37]. Water is a universal solvent, used to extract plant products with antimicrobial activity [38]. All accessions showed quite good extraction yield in water. Yield of aqueous extract varied from 3.0 to 4.1 g of 100 g dried leaf mass used. Maximum yield was obtained from Punjab accession and least from MP accession. The phytochemical analyses of the plants are very important socially as well as commercially. It has great interest of pharmaceutical sector to produce new and innovative drugs for the treatment of various pathogens and diseases. Conventionally, crude plant extracts are used by populations all over the globe for medicinal and therapeutical purposes. Their bioactive chemical components mediate beneficial responses often, although their efficacy and mechanisms of action have not been tested scientifically in majority of the cases [39]. Quality evaluation of herbal preparation is a fundamental requirement of industry and other organization dealing with ayurvedic and herbal products. Our study showed the presence of various medicinally important phytoconstituents from different Aloe vera accessions in good amount, and hence rationalizes the use of plant as an herbal remedy. Quantitative phytochemical estimation of Aloe vera indicates good amounts of alkaloid, phenol and flavonoid contents present in the plant. These are well-known biologically active secondary metabolites, which assist several important medicinal plant properties of Aloe vera [40]. Alkaloids which are one of the largest groups of phytochemicals in plants have amazing effects on humans and used to develop powerful pain killers, toxins against cells of foreign organisms and to eliminate and reduce the human cancer cell lines [41, 42]. Phenols are also a major group of compounds acting as primary antioxidants or free radical scavenger [43]. Flavonoids, another constituent of Aloe vera extract have been proven to perform broad range of biological activities viz. antimicrobial, antioxidant, antiinflammatory, cytostatic, analgesic, anti-allergic, etc. [44]. Different Aloe vera accessions showed that alkaloid contents of the plant are higher than the phenolic and flavonoid contents. According to quantitative screening of Aloe vera, amounts of most of the tested phytochemicals were higher in north Indian plant samples. Linear correlation between phenolic content, alkaloid content, flavonoid content and
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average temperature of plant collection sites by using pearson- correlation method showed significant effect of temperature. There was significant negative correlation reported between phenol and alkaloid contents with average temperature of plant collection sites. It means that rise of temperature reduced the quantity of phenols and alkaloids which may be responsible for reduction in efficacy of samples [31]. Earlier works done by different researchers on phytochemical estimation of Aloe vera also favor our findings [45, 46]. The antimicrobial property of plants is due to the presence of phenolic compounds, saponin, bryophyllin and other secondary metabolites which are reported to be antimicrobial [47-49]. All extracts were most active against E. coli, S. aureus and S. typhi. Previous studies on Aloe vera also stated that Aloe vera extract is highly active against E. coli [50-52]. Maximum antibacterial activity was shown by accessions which belong to highlands. Aloe vera is a cold sensitive plant and during stress more phytochemicals are produced in plants to withstand the adverse conditions. Studies conducted on plants in stress conditions showed higher production of flavonoids, anthocyanins and mucilaginous substances in stress condition [53]. Our study also emphasizes that lower temperature leads to higher production of alkaloids and phenols, the antibacterial compounds present in Aloe vera. Previous studies on Aloe vera leaf extracts showed that extracts were more effective against gram positive bacteria than gram negative becteria [54, 55]. In the present study, aqueous leaf extract showed good activity against gram positive S. aureus as well as gram negative E. coli and S. typhi. Coopoosamy and Magwa reported that pure compound aloe-emodin and aloin are effective in controlling both gram positive and negative bacteria. Aloe vera is rich in these anthraquinones [56]. Although all extracts were quite effective against all the tested strains but variations have been reported in different climatic zones. Variations in the sensitivity to the bacterial species tested from the plant extracts might be due to different quantity and quality of phytochemical composition in diverse accessions according to influence of their local environmental factors. Compared to our previous finding on antibacterial evaluation of same samples, methanol showed strong potential as a solvent than water [51]. But aqueous extracts are preferred over organic solvents as the latter confers some level of toxicity [57]. This study is most comprehensive attempt to assess the antimicrobial activity of whole leaf aqueous extracts of Aloe vera from different climatic zones of India. It emphasizes on effect of climate on production of antibacterial compounds. It will help in commercialization of plant in particular areas with desired pharmacological effects. But, there is still a great need to investigate many other important aspects which correlate with the plant Aloe vera and its antibacterial potential. CONCLUSION The present study concludes that north Indian Aloe vera samples have higher antibacterial activity as compared to South Indian samples. It was also noted that northern Indian samples have higher content of phenols, alkaloids and
Phytochemical Screening and Antibacterial Activity of Aloe vera Collected
flavonoids. Diverse climatic conditions are responsible for the variations in the quality and quantity of phyto-constituents of different Aloe vera samples. Temperature seems to play a major role for the variations observed in phenols, alkaloids, flavonoids and antibacterial activity in samples collected from different climatic zones. This study may help in selection and to formulate new and more potent antimicrobial drugs of natural origin.
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AUTHORS' CONTRIBUTIONS Sandeep Kumar: Collection, acquisition of data, analysis and drafting of the manuscript.
[19]
Lalita Budhwar: Conception, design and interpretation of data.
[20]
Amita Yadav: Significant involvement in the interpretation of data and revising the manuscript.
[21]
Manila Yadav: Participated in the design of the study and performed the statistical analysis.
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CONFLICT OF INTEREST
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The authors confirm that this article content has no conflict of interest.
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ACKNOWLEDGEMENTS The research was financially supported by UGC, New Delhi under UGC-BSR and UGC-SAP (No. F.3 20/2012, SAP II). Authors are also thankful to DST, New Delhi for providing infrastructure grant under DST FIST programme. REFERENCES [1] [2] [3] [4] [5] [6]
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Revised: December 05, 2015
Accepted: December 08, 2015
