Studies on Leaf extracts of drug Punarnava by comparing its Antioxidant Potential by Spectrophotometric method

 

R Badmanaban*, Prajapati Milan G, Patel CN, DJ Sen and Panigrah Bibhuranjan

Shri Sarvajanik Pharmacy College, Near Arvind Baug, Mehsana-384001, North Gujarat, India, affiliated to Hemchandracharya North Gujarat University, Patan, and North Gujarat, India.

 *Corresponding Author E-mail: badu1977@gmail.com

 

ABSTRACT:

These two plants namely Boerhaavia diffusa L. (Nyctaginaceae) and Trianthema portulacastrum L. (Aizeaceae) commonly called as “Punarnava” they were completely different biological source but used for the same purpose since ancient time. So that it is considered as a source of controversial drug. Here we have given emphasis in our study to compare these two sources by means of their antioxidant potential. In the preliminary study, aqueous and methanolic extracts were prepared in the form of hot decoction revealed the presence of tannins and flavonoid, active constituents responsible for antioxidant properties. Then the concepts were considered to under taken to quantify the variation content. To fulfil its timely needed thrust in research area by adopting modern analytical way for standardization. The active constituents were determined by Folin-Denis method for total Phenolic content and AlCl3 colour complex method for flavanoids. Its antioxidant property was evaluated by DPPH radical scavenging assay, Hydrogen Peroxide Scavenging activity and Nitric Oxide scavenging assay. On the basis of the results of this study Boerhaavia diffusa leaf extracts showed that the potent antioxidant potential when compared to Trianthema portulacastrum leaf extracts against free radicals using specific in vitro models.

 

KEYWORDS: Boerhaavia diffusa, Trianthema portulacastrum, Punarnava, controversial drug, Antioxidant, DPPH, scavenging assay

 


INTRODUCTION:

Controversial drugs are those having completely different biological source but used for the same purpose since ancient time ‘Punarnava’. Ancient texts mention two forms of the drug Punarnava. They are stated, two have different properties. Red and white flowered species are found which are equated with the two forms of ‘punarnava’1.Boerhaavia diffusa L. belonging to family Nyctaginaceae, syn:Punarnava is red flowered form of punarnava. Boerhaavia erecta L. and B. verticillata (Nyctaginaceae) both are white flowered and are known as ‘swetapunarnava’2. B. diffusa roots have been widely used for the treatment of abdominal pain3, and as an antistress agent. Its root extract has antihepatotoxic4, diuretic5, anti-inflammatory6, antifibrinolytic7, anticonvulsant8, and antibacterial properties9. Trianthema portulacastrum L. belonging to family Aizeaceae is also known as ‘swetapunarnava’10. The plant is used in treatment of edema in the lever and spleen11, uteralgea and cough.

 

It has significant hepatoprotective activity against paracetamol and thio-acetamide intoxication in rats12. An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules.

 

The reactive oxygen species produced in cells include hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and free radicals such as the hydroxyl radical (·OH) and the superoxide anion (O2−.).  Considering the traditional uses of this drug and after total polyphenolic content and total flavonoid determination, leaves were predicted to have antioxidant activity. Hence, the aim of this study was to investigate the antioxidant profiles of leaves of both Boerhaavia diffusa L. and Trianthema portulacastrum L. against free radicals using specific in vitro models and compare their activity. Even though the In vivo studies is required for correlation

 

MATERIAL AND METHODS:

Plant material:

The leaves of Boerhaavia diffusa and Trianthema portulacastrum were collected from local field area of Mehsana district. They were authenticated by Botanist as the red flower variety is Boerhaavia diffusa and the white flower variety has been identified as Trianthema portulacastrum. A samples (SSPC/RES/10/2009) as preserved in the form of  the herbarium, Dept. of Pharmacognosy of Shri Sarvajanik Pharmacy College, Mehsana for the future reference.

 

Preparation of extracts:

Preparation of aqueous extract:

The dried leaves of both the drugs were powdered using mechanical method and powder was passed through the 20 # sieve. 100 gm powder of both drugs was taken in stainless steel vessel and mixed with 1000 ml of distilled water. The mixture was boiled for about 2hours. Mixture was filtered and filtrate was then evaporated on hot plate in porcelain dish and concentrated up to 50ml. The extracts were evaporated to dryness under reduced temp. The weight of the dry extractives of Trianthema and Boerhaavia showed that 1.61 gm and 1.48 gm respectively.

 

Preparation of methanolic extract:

The dried leaves of both the drugs were powdered using mechanical method and resulting powder was passed through the 20 # sieve. 100 gm of powder of each drug was extracted by soxhlet extraction. After that the extract was filtered and filtrate was concentrated up to 50 ml using water bath. The weight of dry extractives of Trianthema and Boerhaavia were 1.82gm and 1.65gm respectively.

 

Estimation of total Phenolic content:

Total Phenolic contents were determined by Folin-Denis method13. The tannin and tannin like compound reduce Phosphotungustomolybdic acid in alkaline solution to produce a highly blue coloured solution. The intensity of the solution is proportional to the amount of tannin and can be estimated against standard tannic acid using calibration curve. 1ml of (500μg/ml) the aqueous and methanolic extract of both the drugs was withdrawn in 10ml volumetric flask separately. To each flask 0.5ml of Folin-Denis reagent and 1ml of Sodium carbonate was added and volume is made up to 10ml with distilled water. The absorbance was measured at 700nm.

 

Estimation of total flavonoid content:

Total flavonoid contents were measured by colorimetric assay14. Methanolic and Aqueous extracts were added to 10ml volumetric flask containing 4ml of water. To the above mixture, 0.3ml of 5% NaNO2 was added. After 5 minutes, 0.3ml of 10% AlCl3 was added. After 6 min, 2ml of 1 M NaOH was added and the total volume was made up to 10ml with distill water. The solution was mixed well and the absorbance was measured against a separately prepared reagent blank at 510 nm. Total flavonoid content was expressed as percentage of Quercetin equivalent.

 

DPPH free radical scavenging activity:15

The free radical scavenging activity was followed by the DPPH method. It’s based on the principle of conversion of DPPH in to 1, 1- Diphenyl-2- Picryl Hydrazine. 0.1 mM solution of DPPH in methanol was prepared and 1.0 ml of this solution was added to 3.0 ml of extract solution in methanol at different concentration (4-100 µg/ml). Thirty minutes later, the absorbance was measured at 517 nm. A blank was prepared without adding extract. Ascorbic acid at various concentrations (1 to100 µg/ml) was used as standard. Lower the absorbance of the reaction mixture indicates higher free radical scavenging activity. The antioxidant activity of the extracts were expressed as IC50 and compared with standard. The IC50 value was defined as the concentration (in µg/ml) of extracts that scavenges the DPPH radicals by 50%.

 

Hydrogen Peroxide scavenging activity:16

A solution of hydrogen peroxide (40mM) was prepared in phosphate buffer (pH 7.4). Different dilutions of tests and standards were added to the hydrogen peroxide solution. Absorbance of hydrogen peroxide at 230nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. Percentage scavenging of hydrogen peroxide of the extracts and standard compounds was calculated using the formula:

 

Where,    A control = absorbance of control

A test = absorbance of test

 

Nitric oxide radical scavenging activity:17

Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reaction as described previously. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide. Sodium nitroprusside (5 mM) in phosphate-buffered saline was mixed with different concentrations of the drugs dissolved in the suitable solvent systems and incubated at 25 °C for 150 min. The samples from the above were reacted with Greiss reagent (1% sulphanilamide, 2% H3PO4 and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromaphore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamme was read at 546 nm and referred to the absorbance of standard solutions of potassium nitrite treated in the same way with Griess reagent. Percentage scavenging of hydrogen peroxide of the extracts and standard compounds was calculated using the formula as mentioned in above procedure.

 

RESULT AND DISCUSSION:

Total Phenolic content:

The result of the total phenolic content is shown in (Graph 1 and 2).The results showed methanolic extract of Boerhaavia diffusa showed highest amount of phenolic content when compared to other extracts. While the aqueous extract of Trianthema portulacastrum has the lowest phenolic content. The lesser total phenolic content of Trianthema portulacastrumcan is related to its weaker antioxidant property.

Graph 1 and 2: Results for Total Phenolic content

 

Total flavonoid content:

The result of the total flavonoid content is shown in (Graph 3 and 4). The results showed methanolic extract of Boerhaavia diffusa is having highest amount of flavonoid content when compared to other extracts. While the aqueous extract of Trianthema portulacastrum has the lowest flavonoid content. The lesser total flavonoid content of Trianthema portulacastrumcan is related to its weaker antioxidant property.

 

Graph 3 and 4: Results for total flavonoid content

 

Note: Aq. indicates that aqueous extract, Me. indicates that Methanolic extract

DPPH free radical scavenging activity:

The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. It illustrates that a decrease in the concentration of DPPH radical due to the scavenging ability of the soluble constituents in the methanolic and aqueous extracts of leaves of Boerhaavia diffusa L. and Trianthema portulacastrum L. The standard ascorbic acid, as a reference compound, presented the highest activity at all concentrations. The IC50 values were found to be 48.76µg/ml, 63.29µg/ml, 82.17µg/ml, 97.89µg/ml and 166.67µg/ml for ascorbic acid, methanolic and aqueous extract of leaves of Boerhaavia diffusa L, methanolic and aqueous extracts of Trianthema portulacastrum respectively. This shows that the methanolic extract of Boerhaavia diffusa possess highest hydrogen donating capabilities and acts as an antioxidant while the other extracts were not having significant antioxidant activity as compared to methanolic extract of Boerhaavia diffusa. (Graph 5)

 

Graph 5: Results for DPPH free radical scavenging activity

 

Hydrogen peroxide scavenging activity:

Hydrogen peroxide only initiates lipid peroxidation weakly19. However, its ability to produce active oxygen species is due to its ability to generate highly reactive hydroxyl radical through the Fenton reaction20. The ability of plant extract to scavenge H2O2 could also reflect its ability to inhibit the formation of hydroxyl radical in vivo. Indirect stimulation of lipid oxidation by superoxide as a result of superoxide and hydrogen peroxide act as precursors of singlet oxygen and hydroxyl radical21. Gallic acid was used as a reference compound, presented the highest activity at all concentrations. The IC50 values were found to be 48.76µg/ml, 63.29µg/ml, 82.17µg/ml, 97.89µg/ml and 166.67µg/ml for ascorbic acid, methanolic and aqueous extract of leaves of Boerhaavia diffusa L, methanolic and aqueous extracts of Trianthema portulacastrum respectively. This shows that the methanolic extract of Boerhaavia diffusa possess highest hydrogen donating capabilities and acts as an antioxidant while the other extracts were not having significant antioxidant activity as compared to methanolic extract of Boerhaavia diffusa (Graph 6)

 

Graph 6: Results for Hydrogen peroxide (H2O2) scavenging activity

 

Nitric oxide radical scavenging activity:

Nitric oxide (NO) is a potent pleiotropic inhibitor of physiological processes such as smooth muscle relaxation, neuronal signalling, inhibition of platelet aggregation and regulation of cell mediated toxicity. It is a diffusible free radical that plays many roles as an effectors molecule in diverse biological systems including neuronal messenger, vasodilatation and antimicrobial and antitumor activities22. The scavenging of NO by the extracts was increased in dose dependent manner. It illustrates that a significant decrease in the NO radical due to the scavenging ability of extracts and ascorbic acid. The Ic50 values were found to be 227.59μg/ml, 306.46μg/ml, 567.82 μg/ml, 877.46μg/ml and 973.82 μg/ml for ascorbic acid, methanolic and aqueous extract of leaves of Boerhaavia diffusa L, methanolic and aqueous extracts of Trianthema portulacastrum respectively. This shows that the methanolic extract of Boerhaavia diffusa possess nitric oxide scavenging activity and acts as an antioxidant while the other extracts were not having significant nitric oxide scavenging activity activity as compared to methanolic extract of Boerhaavia diffusa. Nitric oxide scavenging activity of ascorbic acid was performed, similar to that of the extract. (Graph 7)

 

Graph 7: Results for Nitric oxide (NO) scavenging activity

 

CONCLUSION:

On the basis of the results of this study methanolic leaves extract of Boerhaavia diffusa has significant and potent antioxidant potential when compared to aqueous extract of Boerhaavia diffusa and methanolic, aqueous extract of Trianthema portulacastrum. The various antioxidant mechanisms of B. diffusa extract may be attributed to its strong abilities as a hydrogen donor, scavenger of hydrogen peroxide and scavenger of nitric oxide. In future the In vivo studies are required for complete correlation. But this preliminary study can be an alternative method for the Identification and standardization of these drugs

 

ACKNOWLEDGMENT:

Authors are thankful to Shri Sarvajanik Pharmacy College, Mehsana for providing all the facilities and support to carry out this research work.

 

REFERENCES:

1.            R.Vasudevan Nair, Controversial drug plants, Universities Press, India, 2004:pp178.

2.            http://www.agri-history.org/pdf/Boerhaavia.pdf

3.            Kirtikar, K.R and Basu, B.D., Indian Medicinal Plants, Vol. III, Allahabad, Uttar Pradesh, India, 2nd Edition, 1956:pp2045–2048.

4.            Chandan B, Sharma A and Anand K. Boerhaavia diffusa: A study of its hepatoprotective activity. J. Ethnopharmacology, 1991, 31:299–307.

5.            Rawat A, Mehrotra S, Tripathi S and Shama U. Hepatoprotective activity in punarnava .A popular Indian ethnomedicine. J. Ethnopharmacology l, 1997, 56:61–68.

6.            Gaitonde B, Kulkarni H and Nabar S. Diuretic activity of punarnava (Boerhaavia diffusa). Bulletins of the Haffkine Institute (Bombay, India), 1974, 2:24.

7.            Bhalla T, Gupta M, Sheth P and Bhargava K. Antiinflammatory activity of Boerhaavia diffusa. Ind. J. Physio.and Pharmacology, 1968, 12 :37.

8.            Jain G and Khanna N. Punarnavoside: A new antifibrinolytic agent from Boerhaavia diffusa Linn. Ind. J. Chem, 1989, 28:163–166.

9.            Adesina S. Anticonvulsant properties of the roots of Boerhaavia diffusa. Quarterly J. Crude Drug Research, 1979, 17:84–86.

10.          http://www.hear.org/starr/hiplants/images/slideshow/html/trianthema_portulacastrum.htm

11.          Javed A, Farooqui A and Ahmad S. Trianthema portulacastrum. An herbal drug for the cure of edema. J. Herbs Species and Medicinal Plants, 2000, 7:65-70.

12.          Kumar G, Sharmilabanu G, Vanitha P, Sundararajan M and Pandian M. Hepatoprotective activity of Trianthema portulacastrum against paracetamol and thiacetamide intoxication in albino rats. J. Ethnopharmacol, 2004, 92:37-40.

13.          Jain U and Dixit V. Spectrophotomatric estimation of tannins from chyavanprash. J. Ind. Drugs, 2004, 41:469-472.

14.          Kumar S, Kumar D, Manjusha, Saroha K, Singh N and Vashishtha B. Antioxidant and free radical scavenging potential of Citrullus colocynthis (L.) Schrad methanolic fruit extract. Acta pharma, 2008, 58:215-220.

15.          Brand-Williams W, Cuvelier E and Berset C. New parameter for evaluation of free radical scavenging capacity of polyphenols. lebensm-Wiss Technol,1995, 28 :25-30.

16.          Gulcin I, Alici H and Cesur M, Determination of in vitro antioxidant and radical scavenging activities of propofol. Chem. Pharm. Bull, 2005, 53:281–285.

17.          Chakraborthy G, Free radical scavenging activity of Costus speciosus leaves. Ind. J. Pharmaceutical Edu. and Research, 2009, 43 :96-98.

18.          Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Japanese J.  Nutri, 1986, 44:307-315.

19.          Cohen G and Heikkila R. The generation of hydrogen peroxide, superoxide and hydroxyl radical by P-hydroxyl dopamine, dialuric acid and related cytotoxic agents. J. Biol. Chem, 1974, 249 :2477-2452

20.          Namiki M. Antioxidants / Antimutagens in food. Critical Review of Food Sci. and Nutri, 1990, 29:273-300.

21.          Kellog, E and Fridovrich I. Superoxide, hydrogen peroxide and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. Biol. Chem, 1988, 263:4704-4711.

22.          Hagerman E, Riedl M, Jones A, Sovik N,Ritchard T and Hartzfeld W. High molecular weight plant polyphenolics(tannins) as biological antioxidants. J. Agric. and Food Chem, 1998, 46:1887-1892.                

 

 

 

Received on 08.12.2009       Modified on 12.01.2010

Accepted on 18.02.2010      © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 566-569