Research Articles

2018  |  Vol: 4(5)  |  Issue: 5 (September- October)  |  https://doi.org/10.31024/ajpp.2018.4.5.27
Phytochemical study and Antioxidant property of Hemidesmus indicus (L) R. Br. roots

Jayalakshmi B.1*, Kruthika L.1, Amruthesh K. N.2

1Post Graduate Department of Botany, Maharani’s Science College for Women, Mysore-570 006, Karnataka, India

2Applied Plant Pathology Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka, India

*Address for Corresponding Author

Jayalakshmi B.

Post Graduate Department of Botany, Maharani’s Science College for Women, Mysore-570 006, Karnataka, India


Abstract

Objective: Oxidative stress is a process due to generation of free radicals which trigger cell damage. Antioxidants significantly delay or prevent oxidation of oxidizable substrates.  Plants have long been a source of exogenous antioxidants. In this contest the present work to investigate antioxidants properties and phytochemical constituents of  Hemidesmus indicus a very important medicinal plant. Material and methods: Different solvent extracts namely petroleum ether, ethyl alcohol and methanol were prepared .These solvent extracts were subjected to Antioxidant assay by DPPH radical scavenging assay, Hydroxyl radical scavenging assay and Nitric oxide scavenging assay. Phytochemical analysis of petroleum ether, methanol, and alcohol extracts was carried  out  for  the  detection  of active  secondary  metabolite or different constitutes such as tannins, alkaloids, flavonoids, terpenoids, steroids, carbohydrates, protein  and saponins. Results and conclusion: The evaluation of DPPH radical scavenging activity ranged from 25- 34µg/ml. Hydroxyl radical scavenging showed that the methanol and ethanol extract having moderate scavenging activity (32-33 µg/ml) compare to the petroleum ether extract. Nitric oxide radical scavenging activity showed the methanol showed good scavenging activity with IC50 values of 46µg/ml. Hence   lower  the  IC50  value,  higher  will  be  the  scavenging  activity. 

Keywords: Antioxidant activity, Hydroxyl radical scavenging, Nitric oxide scavenging, DPPH radical scavenging, Hemidesmus indicus 


Introduction                        

Free radicals can cause “oxidative stress”, a process that can trigger cell damage. Oxidative stress reflects an imbalance between the systemic manifestation of ROS and a biological system’s ability to readily detoxify the reactive intermediates or result of an imbalance in pro-oxidant/antioxidant homeostasis that leads to the generation of toxic reactive oxygen species, such as hydrogen peroxide, organic hydro peroxide, nitric oxide (Adamu et al., 2005). Antioxidants significantly delay or prevent oxidation of oxidizable substrates when present at lower concentrations than the substrate. Plants have long been a source of exogenous   antioxidants. It is believed that two-thirds of the world's plant species have medicinal properties, and almost all of these have excellent antioxidant potential (Cowan, 1999). The antioxidant potential of plants has received a great deal of attention because increased oxidative stress has been identified as a major causative factor in the development and  progression of several life threatening diseases, including neurodegenerative and cardiovascular disease. Moreover, some plants are considered as important source of nutrition and as a result of that they are recommended for their therapeutic values. In this contest the present work to investigate antioxidants properties and phytochemical constituents of Hemidesmus indicus a very important medicinal plant. It is commonly known as Indian sarsaparilla, it is found in South Asia. It belongs to the family Apocyanaceae It is a slender, laticiferous, twining, sometimes prostrate or semi-erect shrub. Roots are woody, slender, and aromatic. The stem and branches of H. indicus twine anticlockwise and are profusely laticiferous, elongate, narrow and deep purple or purplish brown colour with the surface slightly ridged at the nodes (The Wealth of India, 2005).  H. indicus is used to make beverages and also used in traditional medicines. The extracts from the root are used as a coolant and a blood –purifier. The root is also administered in the fourth and ninth month of pregnancy to prevent miscarriage. They also claim its efficacy in treating ulcers, fever, loss of appetite, gastric, anorexia nervosa cough, excessive Menorrhagia, Diarrhea and Diabetes. It also help in increasing semen count, neutralizes poisons, works as a diuretic and emetic and has anti-inflammatory properties (Udayan and Balachandran, 2009).

Materials and methods

Collection and extraction  of plant material

Roots of Hemidesmus indicus were purchased from the local market pansari shop (Figure 1). Thoroughly washed sample of the test plant was dried in shade and powdered using a waring   blender. Solvent extraction was carried out using soxhlet apparatus. Different solvents in the following order Petroleum ether, choloroform, ethyl acetate and methanol based on polarity from low to high. The solvent extract was concentrated separately under reduced pressure.

Figure1.  (a) Hemidesmus indicus whole plant (b) Roots  of  H. indicus

 

Phytochemical analysis

Phytochemical analysis of petroleum ether, methanol, and alcohol extracts was carried out for the detection of active secondary metabolite or different constitutes such as tannins,  alkaloids, flavonoids, terpenoids, steroids, carbohydrates, protein and saponins. The dried extracts obtained by soxhlet extraction were reconstituted in methanol and 1ml of each extract was subjected to standard phytochemical analysis according to the procedure described by Harborne (1998).

Antioxidant assay

DPPH radical scavenging assay

Free radical scavenging activity of the different extract were measured in terms of  hydrogen donating or radical scavenging ability using stable radical DPPH as described by Blois method (Blois, 1958). 3ml of methanol is taken in all test tubes. Different concentrations of solution (20, 40, 60, 80, and 100µg) were made using stock solutions (0.01g of extract dissolved in 1ml of methanol. Standard ascorbic acid solution was prepared by dissolving o.oo1g of ascorbic acid in 1ml of methanol. For negative standard solution 3ml of methanol was taken in another test tube. Then 1ml of DPPH was added to all the solutions. The mixture was vigorously shaken. Now all the mixture was kept for incubation in dark for 1hour. After incubation absorbance was measured at 517nm for all the mixtures including negative control and standard ascorbic acid solutions. Each experiment was run in triplicates and values are expressed as the mean ± standard deviation (SD). The DPPH scavenging ability was calculated using the following formula.

 Where, Acontrol: absorbance of   the negative control

               ASample: absorbance of the sample

Hydroxyl radical scavenging assay

Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity. The scavenging ability of the compounds was determined according to the method Ruch et al. (1948). This method involves in vitro generation of hydroxyl radicals through Fenton system (Fe2+/Ascorbate/ EDTA /H2O2). For this purpose, the stock solution was prepared by dissolving 0.001g of extract in 1ml of methanol. Reaction mixture was prepared using 0.36ml of 2-Deoxyribose (2.8Mm), 0.33ml of KH2PO4 buffer (20Mm PH -7.4), 0.6ml of 30% H2O2 (1.0Mm), 0.1ml ascorbic acid (100Mm). To this previously prepared FeCl3 –EDTA mixture [0.01ml Fecl3 (100Mm) + 0.1ml EDTA (100Mm) was added. Then to this mixture, stock solution was added in various concentrations such as 20, 40, 60, 80 and 100µl. This solution was incubated at 37°C for one hour. After incubation, 1ml of cold TCA (2.8%) and 1ml of TBA (1%) were added solutions of all the concentrations. Then the mixtures were heated at 100°C for 20 minutes to develop color. After development of color, the solutions were cooled and the absorbance was measured at 532nm. Each experiment was run in triplicates and values are expressed as the mean ± standard deviation (SD). The hydroxyl radical scavenging capacity was calculated using fallowing formula:

Where, Acontrol  = in the absence of FeCl3 – EDTA  and tested compound

              Ablank = in the absence of tested compound

Nitric oxide scavenging assay

The nitric oxide scavenging capacity of the compound is determined according to Green et al. (1982).  The assay is based on generation of nitric oxide (NO) from sodium nitroprusside (SNP) and was measured by Griess reagent. Sodium nitroprusside in aqueous solution at physiological PH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions which can be qualified by Griess reagent. Initially Griess reagent has to be prepared. For this purpose, 0.05g of Napthyl ethylene diamine (0.1%) was dissolved in water. Both the solutions were mixed and made up to 50ml by using distilled water. The 1.5ml of sodium nitroprusside (5mM) in  phosphate buffer  (pH=7.0)  was  mixed  with  various concentrations (20, 40, 60, 80, and 100µl)  of 1ml extract and the mixture was incubated at 25°C for  30min. After incubation, 1.5 Griess reagent was added to the incubated mixture.  The reaction mixture was incubated again at room temperature for 10-15 min. The absorbance of the solutions was measured at 546nm. Each experiment was run in triplicates and values are expressed as the mean ± standard deviation (SD). The nitric oxide scavenging percent was calculated using the following formula:

Where, Acontrol = Absorbance of tested compound in absence of sample of the extract

ASample= In presence of sample of the extract

Statistical Analysis

Statistical calculations like Mean and standard error were carried out using one way ANOVA.

Results

Yield of Extracts

Among the solvent extracts maximum yield was obtained by methanol extract (45.2 g/kg) followed by Ethanol (18.0g/Kg) and Petroleum ether (13.7 g/kg).

Antioxidant activity of Hemidesmus indicus

Free radical scavenging activity was evaluated by hydrogen donating ability using stable radical DPPH. The ability of the extracts to neutralize hydroxyl radical was expressed as 50% inhibitory concentration (IC50) in µg/ml. The Methanol and ethanol extract of Hemidesmusindicus was most active with IC50 value at 25µg/ml and 29µg/ml respectively. Above two extracts showed higher radical scavenging activity compared to standard ascorbic acid (27µg/ml). In hydroxyl  radical scavenging  assay,  hydroxyl radical  generated   through   Fenton system in aqueous media  inhibited  by the scavenging  activity  of  the  extract which  is  expressed  as  IC50 values and  compared with the standard ascorbic acid. The antioxidant activity of Hemidesmus indicus was medium in methanol and ethanol extract with IC50 values 32µg/ml and 33µg/ml. Petroleum ether extract showed a least active with IC50 value at 41µg/ml. So all the extracts showed medium antioxidant activity compared to standard   ascorbic activity (27µg/ml). The formation of nitrite by the reaction of sodium nitroprusside with oxygen is inhibited by scavenging activity of the extract which is expressed as IC50 values and compared with standard ascorbic acid. The methanol and alcohol extracts showed  moderate antioxidant activity with IC50 values at 46µg/ml and 52µg/ml respectively  and  petroleum  ether showed  least activity  with  IC50 value  at 64µg/ml.  Hence lower the  IC50  value,  higher  will  be   the  scavenging  activity (Table 1).

Table1. IC50 value  of DPPH, hydrogen  peroxide  and  nitric  oxide  radical  scavenging  by  different  of Hemidesmus indicus

Solvent  extract

             IC50 (µg/ml)

DPPH

H2O2

  NO

Petroleum  ether

 34±0.63

41±0.64

64±0.4

Methanol

25±0.62

32±0.5

46±0.6

Alcohol

29±0.8

33±0.7

52±0.8

Ascorbic acid

27±0.6

27±0.5

27±0.6

The antioxidant effects of Hemidesmus indicus by DPPH, hydrogen peroxide and nitric  oxide  radical  scavenging  methods  increases  with  increase  in  concentration and  the  data  are  depicted  in figure 2, 3 and 4, respectively.

Figure 2. DPPH  radical  scavenging  of  different  extracts   of  H.indicus   and  standard  ascorbic  acid   at  different  concentration (20Mg-100Mg)

 

Figure 3. Hydroxy   radical  scavenging  of  different  extracts of   H.indicus and  standard  ascorbic  acid  at  different  concentration (20Mg-100Mg)

 

Figure 4. Scavenging  effect  of  nitric  oxide  radical  scavenging of  different  extracts    of  H. indicus  and  standard  ascorbic  acid  at  different concentration(20Mg-100Mg)

 

Phytochemical studies

Petroleum ether showed presence of flavonoids, terpenoids, tannins and proteins. Ethanol   extracts showed the presence of flavonoids, tannins, carbohydrates, and saponins. Methanol  extract  showed  the  presence  of flavonoids,  terpenoids,  tannins,  steroids,  carbohydrates,  proteins  and  saponins (Table 2).

Table 2.  Phytochemical  composition  of  solvent  extracts  of  H. indicus

Phytochemical  compounds

Petroleum ether extracts

Ethyl acetate  extracts

Methanol  extracts

Flavonoids

   +

   +

   +

Terpenoids

   +

    -

 +

Tannins

   +

    +

   +

Steroids

   +

    -

+

Carbohydrates

    -

    +

   +

Proteins

   +

    +

   +

saponins

    -

     -

   +

(+) Present   (-) Absent

Discussion

Antioxidants are compounds that hold back the oxidation of essential biological macromolecules by inhibiting the propagation of the oxidizing chain reaction (Mandal et al., 2009). Oxidative  stress  is  initiated by free radicals which  are  generated  constantly and seek  stability   through  electron  pairing  with  biological  molecules  such  as  proteins, lipid and  DNA  of  healthy  human cells  and  can  cause  various   disease. Natural antioxidant is very effective to control the oxidative stress and hence prevent the imitation of disease. These antioxidant are present in  number of  green  leafy  vegetables, free radical scavenging capacity, inhibition of  lipid  peroxidation, metal ion chelating  ability  and  reducing  ability.

DPPH, hydroxyl radical and nitric oxide scavenging methods are selected for the   determination of antioxidant activity in the current study. The scavenging ability of the samples is the measure of antioxidant activity. In  DPPH  method, a  stable  radical is  used   as  a  substrate to  evaluate  the  antioxidant  activity  of H. indicus  extracts. The antioxidant capacity was determined by  the  DPPH  radical   scavenging  activity. It is a stable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecules. In the  presence of hydrogen  donars, DPPH  is  reduced and  a  free  radical  is generated  from  the scavengers. The reaction of DPPH is monitored by   measuring the decrease of the absorbance of its radical at 517nm. The methanol (25µg/ml) and alcohol (29µg/ml) extract of H. indicus have   medium scavenging activity.

The  hydroxyl  radical  is  the  most  reactive  radical  known  to  initiate  lipid  peroxidation   and  damage of biochemicals (Ravikiran et al., 2016). The scavenging effect of the extracts of hydroxyl radicals generated by Fenton’s system is quantified spectrophotometrically  at  532nm. In  methanol   and  alcohol  extract   of  H. indicus  showed  a   good   scavenging  activity  ,while   petroleum  ether  showed  a  moderate   scavenging activity.

  Nitric  oxide  radical  scavenging  activity  was  determined  by  the  abitity  of  the   extract  to  inhibit the  formation  of  nitric  ions  that can  be  produced   by  the  interaction  of  nitric  oxide   generated  by  sodium  nitroprusside  in   aqueous  solvent  at   physiological  PH (Kumara  et  al.,  2013). The reduced nitrite ions are quantified spectrophotometrically at  540nm.  Hemidesmus  indicus    have  showed  medium  scavenging  activity  with  least  IC50  values.

Petroleum  ether  extracts  have  exhibited  good  inhibition  of  free  radicals  generated  by DPPH,  hydrogen  peroxide  and  nitric oxide  when compared  to  the standard ascorbic  acid. In  hydroxyl radical scavenging  shows  the  methanol  and  ethanol  having  moderate  activity compare  to the  petroleum  ether. In nitric oxide shows all the three  extracts  have  moderate  scavenging  activity.

Methanolic extract  by  DPPH  assay method  shows  appreciable  activity  comparable   to  standard  ascorbic  acid , which  contains  large  amounts of  flavonoids  and phenolic compounds  exhibits  reasonable  antioxidant  and free  radical scavenging  activities (Mandal  et al., 2009). There  is scanty  reports  on  antioxidant of  H. indicus but  in  the  present  shows  methanol  and  alcohol  extract  shows the  good  scavenging  activity  compare  to the   petroleum  ether  extract.

Phytochemical analysis of  the  solvent  extracts  of  H. indicus  roots   reveled that flavonoids  and  tannins  were commonly  present in all the  studied  extracts  expect  alcohol extract. The ethanolic extract of H. indicus, which contains all the phytochemicals that were investigated like  alkaloids , flavonoids, tannins, steroids, and phenols  which was also reported by Nogat et al., (2016). Phytochemical investigations revealed that roots of H. indicus showed  presence of  flavonoids coumarino-ligans and tannins (Baheti et  al., 2006).  Literature reports antioxidant   property of aqueous, methanol and alcoholic extracts. But in the  present  study  three  solvents like  petroleum  ether  ,  methanol, and  alcohol  extracts  were  used and all  of  them  have  shown  good  result.   But  the  activity  of  petroleum   ether  extract   has  not   be   reported  and  the  present  study  confirms  one  more  solvent  which  showed  good  activity.

Totally , the scavenging  activities  of  phenolic  substances  might  be  due  to  reactive  hydrogen  donating  ability  of  hydroxyl  substitute, since  phenol  substance  present  in the  extract  are  good  electron  donors  and  may  accelerate  the   conversion  of  hydroxyl   to  water (Mathew   and  Abraham,  2006). These are several methods for the determination of antioxidant activities. The chemical complexity of  extracts which  is  often a mixture  of  group  of  compounds  with  different  functional groups (Karadag  et al., 2009).

Free  radical scavenging  is an important aspect to maintain  good  health   using  antioxidants  The potent antioxidant  activity from plant  source  in  the  present  study  have validated  and  proved  to be good  source  of  plant  based  antioxidant.

Conflicts of interest: None

References 

Adamu HM, Abayeh OJ, Agho MO, Abdullahi AL, Uba A, Dukku HU, Wufem BM. 2005. An ethnobotanical survey of Bauchi State herbal plants and their antimicrobial activity. Journal of Ethnopharmocology, 99:1-4.

Baheti JR, Goyal  RK,  Shah GB. 2006. Hepatoprotective  activity of  H. indicus  R.Br. in rats. Journal of Experimental Biology, 44:399-402

Blois MS. 1958 Antioxidant determinations by the use of a stable free radical. Nature, 181:1199-1200.

Cowan MM. 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12:564-582.

Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JK, Tannenbaum SR. 1982. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Analytical Biochemstry, 126:131–38.

Harborne JB. 1998. Phytochemical Methods, Chapman and Hall Publications, London,   pp 7-8.

Karadag A, Ozcelik B. Saner S. 2009. Review of methods to determine antioxidant capacities. Food Analytical Methods, 2:41-60.

Kumar S, Pooja  M,  Harika K ,  Haswitha  E, Nagabhushanamm H,  Vidyavathi  N.  2013. In vitro  Antioxidant  activities , total  phenolics  and  Flavonoid  content  of whole  plant  of  H. indicus. Research Journal of Microbiolgy, 6:249-251.

Mandal S, Yadav S, Nema RK. 2009. Antioxidants: A review. Journal of  Pharmaceutical and clinical Research, 4:54-58.

Mathew A, 2006. In vitro antioxidant and scavenging effects of Cinnamomum verum leaf extract assayed by different methodologies. Food and Chemical Technology, 44:198-206.

Nogat M, Ehmadi B, Lawrence R, Saani M.  2016  Phytochemical  screening, antioxidant  and  antibacterial  activity  of  active  compound  from  Hemidesmus indicus, International  Journal of Pharmaceutical and Clinical Research, 8:24-27.

Ravikiran  T, Shilpa  S, Praveen   Kumar  N , Sowbhagya  R , Santhosh  Anand, Anupama  S K, Bhagyalakshmi  D. 2016 . Antioxidant  activity of  H. indicus  R.Br, Encapsulated  poly(lactide-co-glycolide) nanoparticles . Food and Chemical Technology, 11: 9-17

Ruch RJ, Cheng SJ, Klaunig JE. 1989. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis, 10:1003-1008.

The Wealth of India. Vol.III, NISCAIR, New Delhi, 2005, Pp. 27 – 28.

Udayan, balachandran. 2009. Medicinal plants of Arya Vaidya Sala Herbal Garden, AryaVaidyaSala, Kottakkal. Pp. 522.

Manuscript Management System
Submit Article Subscribe Most Popular Articles Join as Reviewer Email Alerts Open Access
Our Another Journal
Another Journal