Standardization of Indonesian Traditional Antihypertensive Medicines (Jamu) through the ACE Inhibitor Mechanism

The use of traditional medicines have been widely known from developing countries to developed countries. In some developing countries, traditional medicines are used for health services at the primary level while in developed countries the use of traditional medicines is growing rapidly. The use of traditional medicine in Indonesia has been carried out for centuries but its use is still empirical and its effectiveness and safety have not been supported by scientifically data.1


INTRODUCTION
The use of traditional medicines have been widely known from developing countries to developed countries. In some developing countries, traditional medicines are used for health services at the primary level while in developed countries the use of traditional medicines is growing rapidly. The use of traditional medicine in Indonesia has been carried out for centuries but its use is still empirical and its effectiveness and safety have not been supported by scientifically data. 1 Jamu is one of the Indonesian traditional medicines made from native sources, like roots, bark, flowers, seeds, leaves, and fruits or using ingredients obtained from animals, such as honey, royal jelly, milk and eggs. The use of herbal medicine is still done traditionally, for example in the form of steeping powder or liquid containing all ingredients of plants that make up herbs. 2 Herbal medicine is an option for some people to maintain health because the price is inexpensive, there are no side effects on the body and the raw materials are easy to find.
Prevalence of hypertension in Indonesia based on the results of measurements blood pressure by 25.8%. Most of the cases of hypertension in the community (63,2%) undiagnosed. Basic Health Research Data (Riskesdas) in 2013 showed that 30.4% of households in Indonesia utilize health services traditional, including 49% of houses stairs use traditional medicinal herbs. Meanwhile, Riskesdas in 2010 showed 60% of the population Indonesia was over the age of 15 years stated he once drank jamu, and 90% of them indicated their existence the benefits of drinking jamu. 3 Using traditional drug as part of the treatment of hypertension increasingly increased in the last decade. This matter caused by several factors, especially the prices of traditional medicines considered cheaper with less side effect. 4 The goal of treatment of hypertension with medicinal plants is to treat high blood pressure by correcting the cause according to the philosophy of medicinal plants as constructive medicine, namely repairing/building damaged organs or systems that cause hypertension. Medicinal plants have advantages in the treatment of hypertension because generally, medicinal plants have a function other than managing hypertension as well as treating comorbidities or complications as a result of high blood pressure. 5 One of the drugs used to restore blood pressure in patients with hypertension obtains an Angiotensin Converting Enzymes (ACE) inhibitor. ACE inhibitors are the drugs of choice because, in addition to treating hypertension, they are equally beneficial for cardiovascular diseases, namely congestive heart failure and left ventricular dysfunction, enhancing the function and anatomy of arteries, improving endothelial function, regressing and stabilizing atherosclerotic plaques and in diabetic nephropathy.
Medicines included in ACE inhibitors work by inhibiting the effects of angiotensin II as a vasoconstrictor. The role of ACE inhibitors in hypertension in addition to reducing levels of angiotensin II also increases levels of bradykinin, which contributes as a vasodilator. Vasodilation decreases peripheral vessel resistance, preload, and afterload in the heart, thereby reducing blood pressure. 6 Some medicinal plants are identified to produce antihypertensive effects and work by inhibiting ACE. ACE inhibitors derived from nature have safety and economic value. ACE inhibitors derived from natural product generally comes from groups of compound peptides, proanthocyanidin, terpenoids and tannins. 7,8 The standardization of antihypertensive herbs (jamu) needs to be done to anticipate global competition in the field of jamu and the availability of antihypertensive jamu that are safe, efficient, and scientifically tested. Saintification of jamu will provide a scientific foundation for the use of herbal medicine empirically through research based on health services and community welfare. 9 This study aims to standardization and saintification antihypertensive jamu through inhibition of the Angiotensin-Converting Enzyme to anticipate global competition in the field of herbal medicine and the availability of safe, effective and scientifically tested antihypertensive herbs.

Extraction and identification of the specific and non-specific parameters
The eight (8) antihypertensive herbs (jamu) were extracted with ethanol by maceration then concentrated until a thick extract was obtained. Determination of non-specific parameters includes the examination of ash content, acid insoluble ash content, water content, water-soluble extract content, ethanol-soluble compound, and microbial contamination using the ALT method. Determination of specific parameters includes organoleptic, extract chemical content and identification of infrared spectrum.

In-vitro antioxidant activity assay
Antioxidant activity assay was carried out in a test tube by measuring the absorbance DPPH (H (2,2-diphenyl-1picrylhyrazyl) reagent solution and the extract solution. A total of 1.5 ml extract was added 3 ml DPPH and mixed until homogeneous. This mixture was incubated for 30 minutes, and then the absorption was measured at a wavelength 516 nm. Methanol is used as a blank and Vitamin C as reference drug. Free radical scavenging activity the extract is determined by calculating IC 50 value, which represents the effective concentration required to reduce 50% uptake intensity compared to the reagent solution. IC 50 was calculated of a percent (%) of various absorbance reductions extract concentration using linear regression.

Preliminary test of inhibition of angiotensin-converting enzyme activity
Preliminary Test Inhibition of Angiotensin-Conversion Enzyme Activity included optimum determination of wavelength, temperature, incubation time, and substrate concentration. Determination of the maximum wavelength is carried out by using hypuric acid at a wavelength of 200 -400 nm. The optimum incubation time test was taken out for 15, 30, 60, 90, and 120 minutes. The optimum temperature was determined by incubating the test solution at temperatures of 30, 32, 35, 37, and 40°C. Determination of optimum substrate concentration was performed using Hyppuryl-Histidyl-Leucine (HHL) substrate at concentration of 2, 4, 5, 6, 8, and ten mM.

ACE inhibitory activity assay measurement
The extracts of the antihypertensive jamu were accurately measured to inhibit the specific activity of Angiotensin-Converting Enzyme (ACE). The examination was conducted out using a spectrophotometer under aerobic conditions. The excellent results of the optimization procedure was applied to measure the ACE activity against the extract objectively accurately.
Test solution of 50 µL was gently put into a test tube, then 50 μL of HHL substrate solution was added thoughtfully to the optimum concentration. In addition, pre-incubation of the test solution was carried out at 37°C for 10 minutes. After pre-incubation, 100 µL of the ACE solution is added to the test tube and homogenized using a vortex mixer. The prepared mixture was incubated at optimal temperature and optimum time. Then immediately added 250 µL HCI 1 N to discontinue the reaction. The formed hippuric acid was carefully extracted using 1.5 mL ethyl acetate. The prepared mixture was centrifuged for 10 minutes, and an ethyl acetate layer was gently taken at the top and evaporated at 100°C for 5 minutes. The formed precipitate was dissolved with 3 mL distilled water, and the absorption was precisely measured using a spectrophotometer at a maximum wavelength. Analyses with the similar procedure was carried out on Captopril as a reference drug. Boric acid buffer solution as blanks and control blanks. ACE enzyme solution was added to the blank solution mixture, while the control blanks do not include enzymes. 10,11 Percent inhibition is calculated using the formula A / B x 100%, where A = [absorbance of the blank solution] -absorbance control blank -absorbance of the sample solution; B = [Absorbance of the blank solution -absorbance of the blank control solution] -absorbance of control sample. 12

RESULTS
The composition of plants used in antihypertensive jamu can be seen in Table 1.
The results of measuring non-specific and specific parameters of each extract can be seen in Tables 2 and 3, in-vitro measuring antioxidant activity (IC 50 ) and ACE inhibitor activity (IC 50 ) can be seen in Table 4 and 5.

Results of preliminary test of inhibition of Angiotensin-Converting
Enzyme activity show that the maximum absorption of hippuric acid is at a wavelength of 246 nm. The optimum incubation time remains 90 minutes. The optimum incubation temperature is shown at 37°C, and the optimum substrate concentration is present at eight mM.

DISCUSSION
The utilization of traditional medicines aimed at maintaining health and treating diseases in developing countries is grow rapidly. Indonesia has many medicinal plants that are potentially to be used as traditional prescriptions and have been used for generations. Traditional medicine in Indonesia is known as jamu. 1        tend to control their blood pressure through various methods, one of which is by using antihypertensive herbs. 1,3,13 Therefore, that traditional medicines can be used in health care facilities, and the availability of safe and nutritious herbal medicine needs to be standardized. Standardization of herbal medicine refers to regulations of Indonesia Minister of Health No. 1109 / Menkes / Per / IX / 2007 regarding the administration of effective treatment alternative, complementary health facilities. Standardization represent a critical stage in conducting research and drug development natural medicine in Indonesia to guarantee quality and safety of these drug preparations. In this research, standardization of antihypertensive jamu extract carried out which involves non-specific parameter, example water content, ash content, and acid insoluble ash content, and specific parameters particularly organoleptic, chemical content, and IR chromatogram pattern. 14 Based on the results presented in Table 2, the water content of the herbal extract obtains 5.67-7.92% v/w or less than 10%, so that concluded the eight extracts fulfilled standard specifications. The water content in the extract is barely than 10% minimize the growth of fungi and mold so that the durability and quality of the extract when storage remains good. Ash content measurement is intended to determine the quantity of inorganic material or minerals left after the graying process. The eight herbal extracts have a total ash content of 2.46-7.24%. This value complies with the standard requirements for total ash content, which is equal to no more than 10.2%. The physical properties of an ingredient or extract can be influenced by the levels of inorganic or mineral compounds contained in the extract.
Determination of acid-insoluble ash content aims to determine the amount of ash content obtained from external factors or contamination originating from sand or soil. The Indonesian Ministry of Health requires that the acid insoluble ash level should not be more than 0.7%. The measurement results show that the eight herbal extracts meet the acid insoluble ash content standard. Determination of acidinsoluble ash content is proposed to assess extracts from earth and sand contamination. Determination of the concentration of extract in the solvent water and ethanol is an indicator of the number levels of compounds that can be found. The physical properties of plants determine the amount of compound content that can be dissolved in solutions. The results showed that the herbal extract has the highest solubility in water, which is 45.25 -60.12%. The eighth extracts show more soluble in alcohol solvents, so when formulating the product of the extract can using alcohol solvents. 15 Testing for bacterial contamination is one of the tests to measure the purity of the extract. This test comprises the determination of the number of microorganisms allowed and to indicate the absence of certain bacteria in the extract. The test results showed that the eight extracts were uncontaminated with coliform, mold, and yeast microbes. The maximum limit of microbial contamination in herbal medicines is present 1x10 5 colonies / g while the maximum limit of yeast mold contamination is 1 x 10 -3 colonies/g. 15 Organoleptic was done by observing the physical form of the extract aim as an initial introduction to use the senses. Determination was done by describing the shape and color. Extracts 1, 3, 4, 5, 6, and 7 are yellowish-brown in color and the form of a paste. While extracts 2 and 8 yellowish-brown and liquid. Phytochemical screening aims to find out the existence of groups of secondary metabolites contained in the extract and can also be describe extract content qualitatively.
Phytochemical screening results showed that the extract contained flavonoids, alkaloids, tannins, saponins, quinones and triterpenoids. Infrared Spectrophotometry is a method that can observe the interaction of molecules with electromagnetic radiation that is in the wavelength region of 0.75-1000 μm or at wavenumbers 13,000-10  Table 4 it can be seen that herbal extracts number 1, 4, 7 and eight are classified as very active antioxidants, extracts numbers 2, 3 and five are classified as active antioxidants, and extract number 6 is classified as moderate antioxidants. 17 DPPH is an oxidizing agent that can be a free radical in testing antioxidant activity assay. Using this method is easy, simple, sensitive, fast, and requires a small sample. The test was done by calculating the value of IC 50 , namely the concentration of the test extract that can capture 50% of free radicals obtained through the regression equation.
The smaller the IC 50 value of a test compound, the more effective it is as a free radical antidote. The ability to reduce DPPH radicals in herbal extracts is related to compounds contained in it, namely compounds polyphenols and tannins. Polyphenol compounds and tannins can donate hydrogen. Antioxidant activity of these compounds occurs in the cessation of radical chain reactions that occur. Compounds that have antioxidant activity will react with DPPH through electron administration from antioxidant compounds to DPPH. This reaction causes a decrease in the color intensity of the solution Purple DPPH. The greater the concentration then the intensity of the purple color decreases DPPH that can be measured using absorption UV-VIS spectrophotometer at a wavelength 516 nm. 16,18 The preliminary test of ACE inhibitory activity aims to determine the optimum conditions for enzyme activity so that it can take place optimally on the following sample measurements. Optimization was done because the rate of the reaction catalyzed by enzymes is influenced by several factors, including temperature, pH, and substrate concentration. 19 In the preliminary test, the maximum wavelength is determined using hippuric acid, incubation time, incubation temperature, and substrate concentration to be used at the time of testing. In the preliminary investigation, pH optimization was not carried out because based on the optimal pH incubation literature in the test was 8.3. 20 The higher the absorption is obtained, the more products are produced, so the enzyme activity becomes greater. Based on the preliminary test results of ACE inhibitory activity, optimum conditions were acquired at 246 nm wavelength, the optimum incubation time of 90 minutes, substrate concentration of 8 mM, and incubation temperature of 37°C.
ACE inhibitory activity assay was performed on captopril (as reference drug) and herbal extract sample no. Phaleria macrocarpa is one of Indonesia's native plants which owns medicinal properties. Empirical leaves and fruits have been used to treat various types of illnesses like cancer, liver, heart disease, diabetes, rheumatism, kidney disorders, strokes, and high blood pressure. 24 Mahkota Dewa contains mahkotaside, mangiferin, kaempferol-3-O-d glucoside, dodecanoic acid, palmitic acid, ethyl stearic, and sucrose. 25 Mahkota Dewa additionally exerts an antidiabetic effect which works to inhibit alfa glucosidase and has an antidiabetic effect in rats induced by streptozotocin. 26 Gynura procumbens is used for the treatment of various diseases like hypertension, vasodilators, fever, diabetes mellitus, and hyperlipidemia in Indonesia. 29 G procumbens water extract in rats has anti-hypertensive effects, decreases lactate dehydrogenase levels, creatinine phosphate kinase, and increases nitric oxide. Ethanol extract of G procumbens can reduce triglyceride and serum cholesterol levels in sterptozotosininduced diabetic rats. The antidiabetic effect of G procumbens ethanol extract is comparable to Biguanid. G procumbens increases glucose metabolism via the glycolysis pathway and inhibits endogenous liver glucose production through the gluconeogenesis pathway. 30 Extracts of G procumbens has activity as an ACE inhibitor with an IC50 value of 431.54 ppm in petroleum ether, 227.41 ppm in ethyl acetate and 452.69 in methanol.
Imperata cylindrica roots contain twelve phenolic compounds were isolated, such as flavonoids, simple phenols, phenolic acids, coumarins, and lignans, potassium, cylindrene, and graminone B. 31 Flavonoids possess the effect of inhibiting Angiotensin Converting Enzym. Inhibition of angiotensin production causes a reduction in aldosterone secretion resulting in natriuresis, a decrease in intravascular fluid volume, and a decrease in blood pressure. 32 Potassium obtains an antirenin an enzyme that plays a role in the production of angiotensin. Potassium is additionally an aldosterone inhibitor. Potassium can decrease membrane potential, causing relaxation of vascular smooth muscle. Relaxation of smooth muscle causes the diameter of blood vessels to widen so that blood pressure drops. Cylindrene produces inhibitory activity against the contraction of vascular smooth muscle and Graminone B is a vasodilator agent. 29 The results of a study conducted by Delima and Sari, 2018 showed that the roots of I cylindrica affected lowering blood pressure in healthy subjects. The mean systolic and diastolic blood pressure in subjects who were given boiled I cylindrica roots were significantly lower than blood pressure before being given an infusion (p <0.01). The decoction of the weed root decreases both systolic and diastolic blood pressure in the subject.
Mean systole blood pressure after taking I cylindrica roots infusion (95.33 ± 5.381) mmHg, lower than before drinking infusion of (107.07 ± 5.800) mmHg. The mean diastolic pressure after drinking Imperata root infusion was (64.27 ± 3.693) mmHg, lower than before drinking Imperata root infusion of (70.00 ± 3.854) mmHg. 33 Centella asiatica is an herbal plant of the Mackinlayaceae family. C. asiatica can be found in countries like Indonesia, Sri Lanka, Malaysia, Australia, Iran, Melanesia, New Guinea, and other Asian countries. C. asiatica has chemical content, namely triterpenoids, essential oils, flavonoids, and other components. Triterpenoids are the main component of C. asiatica and become a marker of the quality of C. asiatica. Triterpenes are in the form of asiatic acid, madecassic acid, and their respective glycosides, asiaticoside, and madecassoside. Essential oils such as caryophyllene, farnesol, humulene, and bicyclogerma. Flavonoids in the kind of quercetin, routine catechin, and naringin and other components in the form of polysaccharides, polyene, alkene, fatty acids, sesquiterpenes, alkaloids, sterols, carotenoids, and tannins. 34 Asiatic acid is a triterpenoid that has antioxidant, anti-inflammatory, antihyperlipidemic, and antidiabetic activity in mice induced by Streptozotocin. A study conducted by Manesa et al. 2016 showed that Asiatic acid exerts an antihypertensive effect, improving vascular function through the retention of endothelial nitric oxide synthase (eNOS) and p47phox expression in rats of L-NAME hypertension. Asiatic acid also has ACE inhibitor action by reducing the activity of the angiotensin renin system and increasing vascular function by decreasing sympathetic nerve activity. 35 Bay leaves (Syzygium polyanthum) possesses a variety of pharmacological activities, namely as antihypertensive, antidiabetic, antioxidant, antidiarrheal, anti-inflammatory, immunomodulatory, antibacterial, and anticancer. Active compounds which play a role in its pharmacological activities are quercetin, gallic acid, caffeic acid, and phenolic acid. 36,37 Water and methanol extract of S. polyanthum possess antihypertensive activity in mice, caused by terpenoid phenolic (eugenol), tannins, and flavonoids compounds. Eugenol has vasorelaxant so that it can reduce blood pressure. 38 Antihypertensive effects are thought to inhibit beta-adrenergic and cholinergic receptors through nitrite oxides production. 39 S. polyanthum leaves have ACE inhibitor activity at a concentration of 100 ppm, with the percentage of inhibition is 53.37%. 40 S. polyanthum leaves extract causes vasorelaxation and inhibits Angiotensin-Converting Enzyme (ACE), thereby reducing elevated blood pressure. Studies conducted by Ismail and Wan Ahmad show that water and methanol extracts of S. polyanthum cause vasorelaxation in WKY and SHR mice. 39