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ORIGINAL ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 1  |  Page : 2-6  

Ultraviolet spectrophotometric method: Not possible for the simultaneous estimation of active constituents in Phyllanthus amarus


Oriental College of Pharmacy and Research, Oriental University, Sanwer Road, Jakhya, Opposite Revati Range Gate No. 1, Indore, Madhya Pradesh, India

Date of Web Publication21-Apr-2017

Correspondence Address:
Rakhi Khabiya
Oriental College of Pharmacy and Research, Oriental University, Sanwer Road, Jakhya, Opposite Revati Range Gate No. 1, Indore, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-9234.204909

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   Abstract 

Objective: Our objective was to develop and validate UV spectrophotometric method for simultaneous estimation of phyllanthin, hypophyllanthin, and niranthin in Phyllanthus amarus. P. amarus is highly valued in the treatment of liver ailments and has been shown to posses antihepatitis B virus surface antigen activity. Several analytical methods including high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) are reported for respective phytoconstituents. However, there is no simultaneous estimation of phyllanthin, hypophyllanthin, and niranthin, active constituent of P. amarus. Hence, attempts have been made to develop UV spectrophotometric method because of the simplicity and economical advantage of this technique over other analytical techniques as HPTLC, HPLC, etc. Materials and Methods: Standard solution of phyllanthin, hypophyllanthin, and niranthin were prepared in methanol and scanned under a Shimadzu UV spectrophotomer, under full UV (ultraviolet) range (200-400 nm) to get the spectra. Result: After some experiments, it was found that it is impossible to quantify phyllanthin, hypophyllanthin, and niranthin simultaneously by UV spectrophotometric method. This was evident from the UV spectra as they were overlapping with each other. Conclusion: Same class of drugs may have almost the same functional groups, and gradient reverse-phase liquid chromatography and HPTLC will be more useful to separate such complicated mixtures. Therefore, we propose to develop either HPLC or HPTLC method for simultaneous estimation of these compounds.

Keywords: Lignans, method development, phyllanthin, Phyllanthus amarus, validation


How to cite this article:
Khabiya R. Ultraviolet spectrophotometric method: Not possible for the simultaneous estimation of active constituents in Phyllanthus amarus. J Pharm Negative Results 2017;8:2-6

How to cite this URL:
Khabiya R. Ultraviolet spectrophotometric method: Not possible for the simultaneous estimation of active constituents in Phyllanthus amarus. J Pharm Negative Results [serial online] 2017 [cited 2017 Jul 23];8:2-6. Available from: http://www.pnrjournal.com/text.asp?2017/8/1/2/204909


   Introduction Top


Phyllanthus amarus Schum. and Thonn. is a medicinal herb which have been used in Indian System of Medicine for over 2000 years in treatment of dropsy, intermittent fever, jaundice, diarrhea, scabies, ulcer, and wounds,[1] diseases of the cardiovascular, hepatic, gastrointestinal, and urinogenital tract system.[2] It has been mainly used as an hepatoprotective agent[3] in the treatment of liver diseases and disorders, such as hepatitis, jaundice, and liver cancer and has gained the attention of scientist due to its novel antiviral activity.[4] P. amarus has wide variety of pharmacological activity, such as antiamnesic, antibacterial, anticancer, antidiarrhoeal, gastroprotective and antiulcer, antifungal, analgesic, anti-inflammatory, antiallodynic and antioedematogenic, antinociceptic, antioxidant, antiplasmodial, antiviral, aphrodisiac, contraceptive, diuretic and antihypertentive, hepatoprotective, hypoglycemic and hypocholesterolemic, immunomodulatory, nephroprotective, radioprotective effect, and spasmolytic activity.[5] The major chemical constituents are phyllanthin, hypophyllanthin, niranthin, phyllanthusin D, amariin, amarulone, amarinic acid, ent-norsecurinine, sobubbialine, epibubbialine, nyrphyllin, and phyllnirurin.[3] The major lignans present in P. amarus are phyllanthin, hypophyllanthin, and niranthin which possess wide variety of biological activities.

The active chemical constituents of P. amarus are phyllanthin, hypophyllanthin, and niranthin. Structures of these three compounds have been depicted in [Figure 1]. Phyllanthin reduced whole brain acetyl cholinesterase activity in the aged mice, diazepam, and scopolamine-induced amnesic mice as compared to piracetam.[6] Antibacterial effect of methanolic extract of whole plant of P. amarus was mainly due to phyllanthin against drug resistant pathogens.[7] Phyllanthin exhibited antioxidative and hepatoprotective activities against CCl4-induced toxicity in HepG2 cell line[8] and in ethanol-treated primary culture of rat hepatocytes.[9] Phyllanthin is more potent as hepatoprotective as compared to extract from P. amarus.[8] Ethanolic extract of P. amarus was found to be more effective hepatoprotective agent as compared to aqueous extract due to higher concentration of phyllanthin.[10]
Figure 1: Chemical structures: (i) Phyllanthin, (ii) hypophyllanthin, and (iii) niranthin

Click here to view


Phyllanthin and hypophyllanthin have hepatoprotective activities against carbon tetrachloride, ethanol, and galactosamine treatment. Phyllanthin and hypophyllanthin have modulating effect on vascular tension independent of endothelium. Both significantly induce aortic relaxation by blockade of Ca2+ entry into vascular smooth muscle cells and inhibition of PE-mediated Ca2+ release from sarcoplasmic reticulum. Phyllanthin is more potent in causing aortic relaxation relative to hypophyllanthin, could be attributed to its less planar molecular structure.[2] A mixture of phyllanthin and hypophyllanthin exhibited antitumor activities against Ehrlich Ascites Carcinoma in Swiss albino mice.[11] Phyllanthin and hypophyllanthin have protective effect in acetic acid induced ulcerative colitis by modulation of endogenous biomarkers and DNA.[12]

Niranthin and phyllanthin were found to be cytotoxic which suggests use of Phyllanthus lignans as multidrug resistance reversing agents.[13] Niranthin inhibited carrageenan and PAF-induced paw oedema and neutrophil influx in mice and also decreased the specific binding of [3H]-PAF in mouse cerebral cortex membranes.[14] Niranthin at the non-cytotoxic concentration of 50 µm suppressed HBsAg, hepatitis B effective antigen and showed the best anti-HBsAg activity.[15]

Some of the analytical methods reported for the qualitative and quantificative analysis of marker compounds from P. amarus are discussed herewith. Mukherjee et al.[16] have developed the marker profile of methanolic extract of P. amarus along with phyllanthin and hypophyllanthin as standards using high-performance thin layer chromatography (HPTLC) for developing quality control protocol. An HPTLC method was described by Sane et al.[17] and Deb and Mandal[18] for simultaneous estimation of bioactive lignans, phyllanthin, and hypophyllanthin from P. amarus. Tripathi et al.[19] have quantified phyllanthin and hypophyllanthin in six Phyllanthus species using HPTLC. Dhalwal et al.[20] reported a simple HPTLC densitometric method for the simultaneous quantification of phyllanthin, hypophyllanthin, gallic acid, and ellagic acid in the whole plant of P. amarus. Srivastava et al.[21] developed HPTLC method using chiral TLC plates for qualitative and quantitative analysis of phyllanthin, hypophyllanthin, niranthin, and nirtetralin in Phyllanthus species. Nayak et al.[22] have quantified phyllanthin from P. amarus using HPTLC. Alvari et al.[23] have reported a rapid RP-HPLC technique for the determination of phyllanthin as bulk and its quantification in P. amarus extract. Murugaiyah and Chan[24] have analyzed four lignans namely phyllanthin, hypophyllanthin, niranthin, and phytetralin from Phyllanthus niruri L. in plasma using a simple high-performance liquid chromatographic (HPLC) method with fluorescence detection. Wang and Lee[25] have analyzed and identified lignans in Phyllanthus urinaria by HPLC-SPE-NMR. Shanker et al.[26] have developed method for simultaneous analysis of six bioactive lignans in Phyllanthus species by reversed phase hyphenated HPLC technique.

Method for simultaneous estimation of phyllanthin, hypophyllanthin, and niranthin has not been reported by UV spectrophotometric method. However, reports are there for resolution of phyllanthin hypophyllanthin, niranthin, and nirtetralin using chiral TLC plates but the method suffer from poor resolution and also involves the step of derivitization; which leads to poor robustness and stability of the chromophore developed after derivitization. Moreover, chiral plates are costlier. Thus, the proposed method is easy, accurate, simple, cost effective, and less time consuming. It also reduces the time required for switch over of chromatographic conditions, equilibration of the column, and post-column flushing that are typically associated when different formulations are analyzed by HPLC.

Hence, attempt has been made to develop and validate UV spectrophotometric method for simultaneous estimation of phyllanthin, hypophyllanthin, and niranthin in methanolic extract of whole plant of P. amarus. However, soon we found that it is not possible to develop a simultaneous spectrophotometric method for these compounds. The reasons and the outline of the possible method are described in this paper.


   Materials and Methods Top


Chemicals and reagents

All chemicals used were of analytical grade and were purchased from Qualigens Fine Chemicals, Mumbai, India and Spectrochem Pvt. Ltd., Mumbai, India.

Phyllanthin (purity: 99.51% w/w), hypophyllanthin (purity: 99.19% w/w), and niranthin (purity: 99.51% w/w) were purchased from SPIC Pharmaceutical Pvt. Ltd., Chennai, India.

The UV-visible spectrophotometer (UV-2450, Shimadzu, Japan with spectral bandwidth of 1 nm and wavelength accuracy 0.1 nm) was used for the present study.

For the applicability of the simultaneous equation method, three criteria must be fulfilled.[27] The criteria are:



  1. The λmax of the drugs should have appreciable difference.


  2. Drugs should not interact chemically.


  3. The ratio A2/A1 and ay2/ay1 should lie outside the range of 0.1–2.0.


ax2/ax1 A2/A1.

These criteria are satisfied only when two components are reasonably dissimilar.

The information required was:



  • The absorptivity of the first drug at λ1 and λ2, that is, ax1 and ax2, respectively.


  • The absorptivity of the second drug at λ1 and λ2, that is, ax1 and ax2, respectively.


  • The absorbance of the diluted sample at λ1 and λ2, that is, A1 and A2, respectively.


Preparation of standard solutions of phyllanthin, hypophyllanthin, and niranthin

Stock solution of 200 µg/mL each of phyllanthin, hypophyllanthin, and niranthin were prepared by dissolving 5 mg each of accurately weighed phyllanthin, hypophyllanthin and niranthin in methanol and volume was made upto 25 mL with methanol in individual volumetric flask. 1 mL of each stock solution was transferred to 10 mL volumetric flasks and volume of each was adjusted to 10 mL with methanol, to obtain standard solutions containing 20 µg/mL of phyllanthin, hypophyllanthin, and niranthin.


   Results Top


The standard solutions of phyllanthin, niranthin, and hypophyllanthin each of 20 μg/mL were scanned under the UV region from 200 to 400 nm. [Figure 2] represents an overlay of the spectra of phyllanthin, hypophyllanthin, and niranthin.
Figure 2: Overlay spectra of phyllanthin, hypophyllanthin, and niranthin

Click here to view



   Discussion Top


It is clear from [Figure 2] that it is practically impossible to locate λ1 and λ2 in the case of simultaneous estimation of phyllanthin, hypophyllanthin, and niranthin, since λmax of the drugs does not have appreciable difference, ratios A2/A1 and ay2/ay1 do not lie outside the range of a2/ax1 A2/A1.

0.1-2.0 and the spectra are overlapping. Other analytical techniques, such as HPTLC, HPLC, etc. can be used for the simultaneous estimation of phyllanthin, hypophyllanthin, and niranthin. However, HPTLC has gained widespread acceptance as compared to HPLC and is often used as an alternative to HPLC for the quantification of plant products because of its simplicity, accuracy, cost-effectiveness, and rapidity.


   Conclusion Top


Same class of drugs may have almost the same functional groups, and gradient reverse-phase liquid chromatography and HPTLC will be more useful to separate such complicated mixtures. Therefore, we propose to develop either HPLC or HPTLC method for simultaneous estimation of these compounds.

Acknowledgements

The author is thankful to Oriental College of Pharmacy and Research, Oriental University, Indore for providing the facility in terms of solvents, instruments, and supporting staff for successful completion of the work.

Financial Support and Sponsorship

None

Conflicts of interest

The facilities have been provided by Oriental College of Pharmacy and Research, Oriental University, Indore in terms of solvents, instruments, and supporting staff for successful completion of the work.



 
   References Top

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Inchoo M, Chirdchupunseree M. Pramyothin P. Jianmongkol S, Endothelium-independent effects of phyllanthin and hypophyllanthin on vascular tension. Fitoterapia 2011;82:1231-6.  Back to cited text no. 2
    
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Krithika R, Mohankumar R, Verma RJ, Shrivastav PS, Mohamad IL, Gunasekaran P, Narasimhan S. Isolation, characterization and antioxidative effect of phyllanthin against CCl4-induced toxicity in HepG2 cell line. Chem-Biol Interact 2009;181:351-8.  Back to cited text no. 8
    
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Chirdchupunseree H, Pramyothin P. Protective activity of phyllanthin in ethanol-treated primary culture of rat hepatocytes. J Ethnopharmacol 2010;128:172-6.  Back to cited text no. 9
    
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Yadav NP, Pal A, Shanker K, Bawankule DU, Gupta AK, Darokarm MPS, Khanuja SP. Synergistic effect of silymarin and standardixed extract of Phyllanthus amarus against CCl4-induced hepatotoxicity in Rattus novergicus. Phytomedicine 2008;15:1053-61.  Back to cited text no. 10
    
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Kandhare AD, Ghosh P, Ghule AE, Zambare GN, Bodhankar SL. Protective effect of Phyllanthus amarus by modulation of endogenous biomarkers and DNA damage in acetic acid induced ulcerative colitis: Role of phyllanthin and hypophyllanthin. Apollo Med 2013;10:87-97.  Back to cited text no. 12
    
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Kassuya CA, Silvestre A, JrO Menezes-de-Lima, Marotta DM, Rehder VLG, Calixto JB. Antiinflammatory and antiallodynic actions of the lignin niranthin isolated from Phyllanthus amarus. Eur J Pharmacol 2006;546:182-8.  Back to cited text no. 14
    
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Tripathi AK, Verma RK, Gupta AK, Gupta MM, Khanuja SPS. Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography. Phytochem Anal 2006;17:394-7.  Back to cited text no. 19
    
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Dhalwal K, Biradar YS, Rajani M. High-Performance Thin-Layer Chromatography densitometric method for simultaneous quantitation of phyllanthin, hypophyllanthin, gallic acid, and ellagic acid in Phyllanthus amarus.. J AOAC Int 2006;89:619-24.  Back to cited text no. 20
    
21.
Srivastava V, Singh M, Malasoni R, Shanker K, Verma RK, Gupta MM, Gupta AK, Khanuja SPS. Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method. J Seperation Sci 2008;31:47-55.  Back to cited text no. 21
    
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Murugaiyah V, Chal KL. Analysis of lignans from Phyllanthus niruri L. in plasma using a simple HPLC method with fluorescence detection and its application in a pharmacokinetic study. J Chromaogr B 2007;852:138-44.  Back to cited text no. 24
    
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26.
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    Figures

  [Figure 1], [Figure 2]



 

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