Usharani et al. BMC Complementary and Alternative 2019) 19:97RESEARCH ARTICLEOpen AccessEvaluation of the effects of a standardizedaqueous extract of Phyllanthus emblicafruits on endothelial dysfunction, oxidativestress, systemic inflammation and lipidprofile in subjects with metabolicsyndrome: a randomised, double blind,placebo controlled clinical studyPingali Usharani* , Padma Latha Merugu and Chandrasekhar NutalapatiAbstractBackground: Endothelial dysfunction (ED) has been observed in individuals with metabolic syndrome (MetS) andcontributes to the initiation and progression of atherosclerosis. The primary management of MetS involves lifestylemodifications and treatment of its individual components with drugs all of which have side effects. Thus, it wouldbe of advantageous if natural products would be used as adjuncts or substitutes for conventional drugs. The aimof the present study was to evaluate the effect of standardized aqueous extract of fruits of Phyllanthus emblica(P. emblica) 250 mg and 500 mg twice daily on ED, oxidative stress, systemic inflammation and lipid profile insubjects with MetS.Methods: In this randomised, double-blind, placebo-controlled clinical study endothelial function was measured bycalculating reflection index (RI) using digital plethysmograph. Oxidative stress biomarkers used were nitric oxide (NO),glutathione (GSH) and malondialdehyde (MDA). Systemic inflammation was measured by determining high sensitivityC-reactive protein (hsCRP) and dyslipidemia by lipid profile. ANOVA, paired and unpaired t-test were used. P-value 0.05 was considered statistically significant.Results: Out of 65 screened subjects all 59 enrolled completed the study. P. emblica aqueous extract (PEE), 250 mg and500 mg twice daily dosing, showed significant reduction in mean RI, measure of endothelial function, at 8 and 12weeks (p 0.001) compared to baseline and placebo. Significant mean % change was seen in oxidative stressbiomarkers, NO ( 41.89%, 50.7%), GSH ( 24.31%, 53.22%) and MDA ( 21.02%, 31.44%), and systemicinflammation biomarker, hsCRP ( 39.68%, 53.77%) (p 0.001) at 12 weeks with 250 mg and 500 mg twicedaily dosage respectively. Significant mean % change was also seen at 12 weeks with TC ( 7.71%, 11.11%),HDL-C ( 7.33% 22.16%, p 0.05), LDL-C ( 11.39%, 21.8%) and TG ( 9.81%, 19.22%) respectively with250 mg and 500 mg twice daily (p 0.001). PEE 500 mg twice daily was significantly more efficacious thanthe 250 mg twice daily and placebo. No participant discontinued the study because of adverse events.(Continued on next page)* Correspondence: [email protected] of Pharmacology and Therapeutics, Nizam’s Institute of MedicalSciences, Telangana, India The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication o/1.0/) applies to the data made available in this article, unless otherwise stated.
Usharani et al. BMC Complementary and Alternative Medicine(2019) 19:97Page 2 of 8(Continued from previous page)Conclusions: P.emblica aqueous extract significantly improved endothelial function, oxidative stress, systemicinflammation and lipid profile at both dosages tested, but especially at 500 mg twice daily. Thus, this productmay be used as an adjunct to conventional therapy (lifestyle modification and pharmacological intervention)in the management of metabolic syndrome.Trial registration: This study was registered with Clinical Trials Registry – India (CTRI) with the registrationnumber of CTRI/2017/09/009606. The study was registered retrospectively on 4th September 2017.Keywords: Phyllanthus emblica, Emblica officinalis, Amla, Reflection index, Dyslipidemia, Oxidative stress,Systemic inflammation, Endothelial dysfunctionBackgroundMetabolic syndrome (MetS) is characterized by thecoexistence of multiple interrelated cardiovascular(CV) risk factors in a single individual . The National Cholesterol Education Program’s Adult Treatment Panel III (ATP III) identified six componentsof the MetS: abdominal obesity, insulin resistancewith or without glucose intolerance, atherogenic dyslipidaemia, raised blood pressure, prothromboticstate, and proinflammatory state . MetS affects25% of the world population, and its incidence isgradually increasing .Endothelial dysfunction (ED) is abnormal functioningof the endothelial cells, caused by a decrease in bioavailability of vasodilator substances, particularly nitric oxide(NO), and increase of vasoconstrictor substances. Numerous metabolic abnormalities found in MetS result inendothelial cell dysfunction by affecting NO synthesis ordegradation. The frequent metabolic abnormalities thatmay contribute to ED in patients with MetS are hyperinsulinemia, hyperglycaemia, increase in the levels of fattyacids, C- reactive protein, oxidative stress, triglyceridesand low-density lipoprotein cholesterol (LDL-C), and adecrease in high-density lipoprotein cholesterol(HDL-C) .Both obesity and MetS have been independently associated with increased oxidative and inflammatory stress. Oxidative stress is due to the production of reactiveoxygen species (ROS), increased lipid peroxidation andimpairment of antioxidant enzymatic defences, such assuperoxide dismutase (SOD) or glutathione peroxidase(GPx) or combination of these factors . Lipid peroxidation leads to the formation of malondialdehyde(MDA), and increased levels of MDA are found in obesity and insulin resistance. MDA can induce the increasedexpression of pro-inflammatory cytokines, resulting insystemic stress . C-reactive protein (CRP) is a markerof systemic inflammation and a predictor of future atherosclerotic events . Specifically, high sensitivity C reactive protein (hsCRP) is an independent predictor ofcoronary heart disease .Endothelial dysfunction (ED) is the earliest detectablefunctional disturbance in the natural history of the disease and a powerful predictor of future CV events .Therapeutic interventions aimed at reducing CV riskfactors may improve the endothelial function . Theprimary management of MetS involves lifestyle modifications and treatment of its individual components, whichincludes lipid-lowering drugs, angiotensin-convertingenzyme inhibitors, antidiabetic drugs, antiplatelet andantioxidant agents. However, the currently availablepharmacologic interventions sometimes may not sufficiently improve endothelial function or may have side effects . So, alternative treatments are needed toprevent the development of cardiovascular diseases(CVD) in subjects with MetS.Phllanthus emblica (Emblica officinalis, Amla) fruitsare an important dietary source of polyphenolic compounds, which are low molecular weight hydrolysabletannins. This fruit extract has been reported to exhibithypolipidemic, antidiabetic, anti-inflammatory, and antioxidant properties . It has been reported that thetannoid principles of aqueous extract of Amla are a potent inhibitor of lipid peroxide generation and a scavenger of hydroxyl and superoxide radicals in vitro and invivo .Clinical studies provide evidence that P.emblica extract has significantly improved endothelial function andreduced biomarkers of oxidative stress and systemic inflammation in patients with type 2 diabetes mellitus(T2DM) [14, 15]. As there is a paucity of data on the effect of P.emblica on ED in MetS, the present study wasundertaken to evaluate the effect of a standardized aqueous extract of P.emblica, at 250 mg (PEE250) and 500mg (PEE500) versus placebo twice daily on ED, oxidativestress, systemic inflammation and lipid profile in MetS.MethodsThe present study was prospective, randomised,double-blind and placebo-controlled with a 12-weekduration, conducted in the Department of ClinicalPharmacology and Therapeutics, NIMS, Hyderabad,
Usharani et al. BMC Complementary and Alternative Medicine(2019) 19:97India, after written approval from the Institutional EthicsCommittee. Written informed consent was taken fromall the subjects prior to participation in the study.Subjects included in the study were of either gender,aged 30–68 years, having ED defined as 6% change inreflection index (RI) on post salbutamol challenge testand having MetS according to “The International Diabetes Federation” guidelines, dated 2006 . Exclusioncriteria consisted of subjects with severe uncontrolledhypertension, uncontrolled hyperglycemia, impairedhepatic or renal function, cardiac arrhythmia, chronicobstructive pulmonary disease (COPD), bronchialasthma, history of smoking, chronic alcoholism, malignancy or stroke and any other serious disease requiringactive treatment and treatment with any other herbalsupplements. A total of 65 subjects were screened and59 eligible subjects were randomised by computer generated block randomisation.Study medications: The test products, PEE250 (CAPROS 250) and PEE500 (CAPROS 500) and matching placebocapsules used in the present study were supplied byNatreon Inc., USA. Each capsule of the PEE250 andPEE500 comprised of aqueous extract of the edible fruits ofP. emblica, standardized by high performance liquid chromatography (HPLC) to contain not less than 60% of lowmolecular weight hydrolysable tannins ((LMwHTs), comprising of Emblicanin–A, Emblicanin-B, Pedunculagin andPunigluconin as bioactives. Placebo capsules containedmicrocrystalline cellulose, lactose and magnesium stearateas excipients. The HPLC chromatogram of CAPROS isshown in Fig. 1. The HPLC method used was the company’s In-house Monograph method. Briefly, 50 mg ofP.emblica extract was dissolved in 50 ml of distilled water,and filtered through 0.22 μm syringe filter. The filteredFig. 1 HPLC chromatogram of Capros Page 3 of 8solution (20 μL) was injected into Waters HPLC system(equipped with e2695 separation module, Photodiode Arraydetector (2998), and Empower3 pro Software). Compoundswere separated on a NovaPak RP C18 150 3.9 mm, 4 μ(Waters corporation, WAT086344), column using 0.1 Msodium acetate–acetic acid buffer (pH 3.9) as the mobilephase at the flow rate of 0.6 ml/min and detection wavelength 280 nm. The percentage content of the LMwHTswas calculated using area of the LMwHTs peaks and thelinear regression equation of the external standard.Eligible subjects were enrolled and randomised by theinvestigator. The containers containing drugs were sequentially numbered and were dispensed by the pharmacist to the subjects according to the randomly allocatedsequence so as to receive one capsule of PEE250, PEE500or placebo twice daily for 12 weeks. Subjects returned forfollow up visits at 4, 8 and 12 weeks of therapy, when subjects were evaluated for efficacy and safety. Pharmacodynamic evaluation of endothelial function (RI) wasconducted at every visit. A 10 ml blood sample was collected in plain blood collection tubes after an overnightfast of 12 h for evaluation of NO, MDA, glutathione(GSH), hsCRP and lipid profile at baseline and at the endof 12 weeks of treatment. A complete physical examination and safety lab investigations for hematological, hepatic and renal biochemical parameters were conducted atbaseline and at the end of the study, and as required during the study. Subjects were enquired for the presence ofadverse drug reaction (ADR) at every visit, and any reported ADRs were recorded in the case report form.Compliance with therapy was assessed by pill countmethod.Endothelial function was evaluated by salbutamol challenge test using the digital volume plethysmography
Usharani et al. BMC Complementary and Alternative Medicine(2019) 19:97(DPG) as reported by Chowienczyk et al. and Naidu etal. [16, 17]. Subjects were examined in supine positionafter 5 min of rest. A digital volume pulse (DVP) was obtained using photo plethysmograph (Pulse Trace PCA2,PT200, Micro Medical, Kent, UK) transmitting infra-redlight at 940 nm, placed on the index finger of right hand.The signal from the plethysmograph was digitized usinga 12 bit analogue to digital converter with a samplingfrequency of 100 Hz. DVP waveforms were recordedover 20 s period and the height of the late systolic / earlydiastolic portion of the DVP was expressed as a percentage of the amplitude of the DVP to yield the RI, as perthe procedure described in detail by Millasseau et al.. After DVP recordings had been taken, three measurements of RI were calculated and the mean value wasdetermined. Subjects were then administered 400 μg ofsalbutamol by inhalation. After 15 min, three measurements of RI were obtained again and the difference inmean RI before and after administration of salbutamolwas used for assessing endothelial function. A change of 6% in RI post salbutamol was considered as endothelialdysfunction.A spectrophotometer was used to estimate serumMDA (Thiobarbituric acid reactive substance test), NO(Colorimetric detection with Griess reagents) and GSH(Ellman’s method) levels [19–21].Peripheral venous blood was collected in plain bloodcollection tubes after an overnight fast of 12 h after thelast dose of medication for the estimation of haemoglobin, blood urea, serum creatinine, liver function test,lipid profile using standard techniques.Primary and secondary efficacy parametersThe primary efficacy criterion was more than 6%change in RI (a measure of ED), assessed at 4,8 and 12weeks. Secondary efficacy parameters included improvements in oxidative stress markers - NO, GSH and MDA,systemic inflammation marker hsCRP and the lipid profile at 12 weeks. Safety and tolerab