INTRODUCTION:

Herbal medicine (Jamu) is a traditional Indonesian medicine used for the prevention and treatment of diseases. Jamu, as a cultural heritage, possesses an enormous potential asset for Indonesia. Jamu is one of Indonesia's cultural products based on local resources. Indonesia has around 30,000 species of natural resources globally after Brazil and Zaire, and 7000 are medicinal plants¹. The results of Basic Health Research in 2013 showed that 49% of Indonesian people use herbal medicine to treat their ailments. The use of herbal medicine, therefore significantly, is, however, limited to self-medication and has not been done in health facilities².

Hypertension and diabetes mellitus are related to diseases that can cause atherosclerotic disease and other cardiovascular risks. Hypertension also contributes to diabetic retinopathy, which is a primary cause of blindness. Hypertension and diabetes must be managed and treated appropriately. In diabetic patients, 30% of deaths correspond to hypertension³. The prevalence of hypertension within patients with diabetes mellitus by sex as a whole is 70%, 32% in men, and 45% in women. While the prevalence of hypertension by region is 49% in Indians, 37% in Europeans, and 35% in Asians this data illustrates that hypertension in diabetes mellitus will often be found compared to individuals without diabetes⁴.

Hypertension that is unmanaged properly will accelerate damage to the kidneys and cardiovascular abnormalities^5-6. Blood pressure control will protect against micro and macrovascular complications while managing and controlling hyperglycemia. The pathogenesis of hypertension in patients with diabetes mellitus is very complicated and is influenced by several factors, namely insulin resistance, plasma blood sugar levels, obesity, and other factors in regulating blood pressure regulation^3-7.

One of the causes of diabetes mellitus, primarily type 2, is oxidative stress, which can induce insulin resistance in peripheral tissues and damage insulin secretion from pancreatic beta cells. Besides, hyperglycemia is also involved in the process of free radical formation. Hyperglycemia causes glucose auto-oxidation, protein glycation, and the activation of the polyol metabolic pathway, which further accelerates reactive oxygen species formation. Excessive lipid oxidation can produce radical compounds, so antioxidants are needed to reduce it⁸.

The ideal antihypertensive drug in patients with diabetes mellitus is a drug that can regulate blood pressure, does not interfere with glucose or lipid metabolism, acts as a renoprotective, and reduces death due to cardiovascular complications. Strategi management to prevent the progression of kidney disorders in people with diabetes is managing proteinuria, hypertension, hyperglycemia, other risk factors such as dyslipidemia, and lifestyle changes. Antihypertensive drugs are renoprotective, like ACE inhibitors will reduce blood pressure and protein excretion. This situation will reduce the risk of terminal kidney failure and improve life expectancy⁹. The use of herbal medicine, as part of the treatment of hypertension and diabetes mellitus increased in the last decade due to several factors, especially the price, which is considered more inexpensive with fewer side effects^10-11. A study conducted by Paramita et al., 2017 showed that 70.9% of hypertensive patients at Community Health Centers use indigenous medicines. All patients (n=45) practiced jamu, which were theoretically proven to lower blood pressure. However, indigenous medicines practiced are not included in the criteria for standardized herbal medicines or phytopharmaceuticals. Only 15.2% of patients using jamu are following the Republic of Indonesia Drug and Food Examination Agency regulations on herbal medicine criteria¹².

This study aims to standardize and assay antihypertensive, antidiabetic, and antioxidant activities of Indonesia Traditional Medicine (Jamu) to anticipate global competition in the field of herbal medicine and the availability of safe, real efficacious, and scientifically tested herbs. This research is valuable to provide a scientific basis for the use of antihypertensive herbs that possess antidiabetic, and antioxidant activity, and to provide other data on antihypertensive herbs with ACE inhibitor activity and to represent a start in the search for and development of alternative drugs for antihypertensive and antidiabetic

MATERIALS AND METHODS:

Materials:

Herbal medicine (Jamu) were obtained from herbal Market in Jakarta, Ethanol (Bratachem), Aquadest (Bratachem), Aquademineral (Bratachem), Ethyl Acetate (Merck), DMSO (Merck), Hippuric Acid (Sigma), Captopril (Sigma), HHL (Sigma), ACE from rabbit lung (Sigma), NaOH (Merck), Boric Acid (Sigma), DPPH (Merc), vitamin C (sigma). Α-Glucosidase (Sigma), p-nitrophenyl-α-D-glucopyranoside substrate (Sigma), dimethyl sulfoxide (DMSO) (Merck), Bovine Serum Albumin (BSA) (sigma), Na2CO3 (Merck), buffer phosphate pH 7, acarbose (Sigma), NaCI (Merck).

Extraction and Standardization of antihypertensive jamu:

Jamu was extracted by maceration using ethanol and then concentrated until a thick extract is acquired. Standardization of non-specific parameters includes checking ash content, ash content, which is insoluble in acids, water content, levels of extracts that are soluble in water, soluble compounds in ethanol, and coliform contamination and mold/yeast rates. Standardization of specific parameters includes organoleptic examination, extract chemical content and identification of infrared spectra^13-14.

Assay of ACE inhibitory activity:

Measurements ACE inhibitor activity were made using a spectrophotometer in aerobic conditions. A total of 50μL of the test solution was put into a test tube, then added 50μL of HHL substrate solution and pre-incubated at 37⁰C for 10 minutes. A total of 100μL of ACE enzyme solution was added to the test tube and homogenized with a vortex. The mixture was incubated at 37⁰C for 90 minutes. Then to the solution mixture was added 250μL HCI 1 N to stop the reaction. The formed hippuric acid was extracted using 1.5mL ethyl acetate and centrifuged for 10 minutes. The ethyl acetate layer is taken slowly and evaporated at 100⁰C for 5 minutes. The formed precipitate was dissolved with 3mL aquadest. The absorption is then measured using a spectrophotometer at a wavelength of 246nm. The same procedure was carried out with Captopril as a standard and Boric acid buffer solution as blank solution and blank control. To the blank solution, the ACE enzyme solution was added, while the blank control was unadded to the enzyme solution. Percent inhibition of ACE inhibitor activity wasa calculated using the formula A/B x 100%, where A = [absorbance of empty solution] — absorbance Blank control — absorbance of sample solution; B = [absorbance of a blank solution - absorbance of blank control solution] - absorbance of sample control¹⁵.

Assay of Alpha-glucosidase inhibitory activity:

A total of 10μL extract solution at concentrations of 125, 250, 500, 750, and 1000ppm was added 50μL of phosphate buffer solution pH 7 and 25μL p-nitrophenyl-α-D-glucopyranoside 10mM. Furthermore, the solution was added 25μL enzyme solution concentration of 0.04 U/mL, and incubated at the optimum time and temperature, and incubated at the optimum time and temperature. Then 100μL Na₂CO₃ 200mM was added. The absorbance was measured with a microplate reader at a wavelength of 410nm. The Acarbose was used as reference. Percent inhibition of alpha-glucosidase inhibitory activity was calculated following the formula:

A₀-A₁,

----------

A₁

where A₀ is the absorbance of the control; A₁ is the absorbance of the sample. IC₅₀is assessed using linear regression by plotting the concentration of percent inhibition¹⁶.

Assessment of antioxidant activity:

A total about 500μL extracts at concentrations of 125, 250, 500, 750 and 1000μg/ml plus 500μL DPPH 0.125 mM. The mixture is then vortexed and incubated for 30 minutes. Besides, this solution is measured absorbance at a wavelength of 517nm. Absorbance measurements were equally taken. The results of the determination of antioxidants compared with vitamin C. The percentage of antioxidant inhibition against free radicals was calculated following the formula Inhibition (%) = (absorbance of blank-absorbance of extract)/absorbance of blank x 100¹⁷.

RESULT:

Standardization results on non-specific parameters:

The results regarding standardization of herbal medicine on non-specific parameters and specific parameters, respectively can be seen in tables 1 and 2.

Table 2. Standardization results on specific parameters

Parameter
Organoleptic (colour and texture)	Chemical content	identification of infrared spectrum
yellowish-brown and paste	Flavonoid, alkaloid, tannin, saponin, steroid, triterpenoid	OH; C-H (aliphatic); C=C; C=O; C-N; C-H (aromatic);

ACE inhibitor, antidiabetic and antioxidants activities:

The results about measurements of the IC50 value of the ACE inhibitor activity, alpha-glucosidase inhibitors, and antioxidants can be seen in table 3.

Table 3. Standardization results on specific parameters

sample	IC₅₀ (µg/ml)
sample	ACE inhibitor	a-glucosidase inhibitor	antioxidant activity
Extract	103,75	49,95	11,4
reference	0.64	0.268	5.13

DISCUSSION:

Herbal medicine has been used by people in Indonesia to prevent and treat various diseases, including hypertension. A national survey conducted by Hussain et al., 2016 showed that 46.4% of Indonesian people were diagnosed with hypertension, but only 9% were obtaining adequate treatment¹⁰. Society tends to treat hypertension by utilizing various methods, including antihypertensive herbal medicine. A study conducted by Rahmawati and Bajorek, 2018 concluded that as many as 68.5% used traditional medicines to lower blood pressure¹⁸. Standardization represents a crucial stage in the development of herbal medicine. Standardization of a drug preparation (extract or simplicia) obtains a necessity to create pharmaceutical and therapeutic quality reproducibility¹⁹. Standardization consists of two criteria, namely non-specific and specific parameters. Standardization on non-specific parameters focuses on chemical, microbiological, and physical aspects that will affect consumer safety and stability, including water content, heavy metal contamination, and aflatoxin. In contrast, standardization on specific parameters focuses on compounds or groups of compounds responsible for pharmacological activities. The chemical evaluation involved is intended for the active compound's qualitative and quantitative analysis^20-21.

Table 1 shows that the extract meets the requirements of non-specific parameters. The extract has water content (5.85% w/v), ash content (5.92% w/w), acid insoluble ash content (0.41% w/v), alcohol-soluble extract content (60.16%), water-soluble extract (31.93%), the extract was not contaminated with coliforms and negative yeast and mold rates.for non-specific parameters, namely moisture content (5.85% w/v), ash content (5.92% w/w), acid insoluble ash content (0.41% w/v), extract content soluble in alcohol (60.16%), water-soluble extract content (31.93%), the extract was not contaminated with coliforms and negative yeast and mold rates. The organoleptic examination is an initial introduction using the eye senses by describing the shape, color, smell, and taste carried out by observing the extract's physical form²⁰. Organoleptic examination results showed that the extract was brownish-yellow in color with a paste form. The water content determination results indicated that the extract was included in the thick extract criteria with a moisture content of 5.85±0.24²². The Indonesian Ministry of Health stipulates that an extract's water content should not exceed 10%¹⁸. The high water content will be a medium for the growth of microorganisms and fungi. The value of water content will be correlated with the number of mold/fungus and coliform contamination. From table 1, it can be seen that the extract is free from coliform and mold/yeast contamination. An extract must have a mold/yeast rate of not more than 104 CFU/gr^23-24. The ash content determination results showed that the herbal extract had an ash content that met the requirements (5.92% w/w). Based on the Decree of the Minister of Health of the Republic of Indonesia Number 261 / MENKES / SK / IV/2009, the extract's ash content should not be more than 10.2%. The ash content's determination aims to provide an overview of the internal and external mineral content²⁵. Total ash content parameters relate to the purity and contamination of an extract²⁴.

Measurement of acid-insoluble ash content aims to determine the amount of ash originating from external factors, originating from impurities originating from sand or silicate soil^26-27. The acid-insoluble ash content in the herbal extract was 0.41 w/v. This level fulfills the requirements, which is less than 0.9%²⁸. Water and ethanol-soluble extract content is used to estimate the active substance content based on a compound's polarity. The results of this examination can provide an overview of compounds that are polar, semi-polar, and non-polar. The total value of the soluble extract content in water and ethanol should not be more than 100%. The level of active substances in a simplicia is influenced by plant age, harvest time, and the climate in which it grows²⁶. The water-soluble and ethanol content of the herbal extracts were 31.93% and 60.16%, respectively. This value meets the requirements set.

The microbial contamination test is carried out to assure that the extract must not contain pathogenic microbes and does not contain non-pathogenic microbes that exceed the maximum established limit because it affects the extract's stability and is harmful to health²⁷. The results of the coliform contamination examination and the mold/yeast levels in the extract were negative.

The chemical content examination aims to determine secondary metabolite compounds contained in the extract. The herbal medicine extracts' chemical content showed that the extract contained flavonoids, alkaloids, tannins, saponins, steroids, and triterpenoids. Infrared spectrum examination results show that the herbal extract has the OH functional group; C-H (aliphatic); C = C; C = O; C-N; C-H (aromatic).

Table 3 shows the results of examining the extract activity in inhibiting the angiotensin-converting enzyme, inhibiting alpha-glucosidase, and antioxidant activity with IC₅₀ values of 103.75µg/ml, 49.95µg/ml, and 11.4 µg/ml, respectively. The IC₅₀ values of captopril, acarbose, and vitamin C were 0.64µg/ml, 0.268µg/ml, and 5.13µg/ml. ACE inhibitory activity testing consisted of measuring herbal extracts' inhibition against the ACE activity, measuring control samples, blanks, and control blanks carried out spectrophotometrically. Measurement of control samples is carried out as a correction factor if the tested extract provides the resulting absorption at the maximum wavelength of measurement. Testing of extract inhibition against ACE activity was carried out using various concentrations. This variable concentration test was carried out to see the effect of increasing the extract concentration on increasing inhibition. The increasing concentrations of the extracts used were 125, 250, 500, 750, and 1000µg/ml. Synthetic ACE inhibitors such as captopril, ramipril, and lisinopril have side effects such as dry cough, hyperkalemia, rash, dizziness, and taste changes. Therefore, ACE inhibitors that come from natural ingredients were developed, either from food or plants. Natural ACE inhibitors are considered to be safer than synthetic ACE inhibitors²⁹. Secondary metabolites produced by plants are natural compounds identified as ACE inhibitors, namely flavonoids, hydrolyzed tannins, xanthones, procyanidins, and caffeoylquinic acid. Several studies have shown that many ACE inhibitors come from plants, but the identification of their active compounds is still minimal^29-30.

The antihypertensive herbs used in this study consisted of simplicia, namely Curcuma xantorrhizae rhizome, Andrographis paniculata herb, Tinospora crispa caulus, Orthosiphon stannous folium, and Phyllanthus niruri folium. The fifth simplicia contained in this antihypertensive herb contributes to the antihypertensive, antidiabetic, and antioxidant effects. Curcuma xantorrhizae (Temu lawak) is a medicinal plant that is widely used as raw material for herbal components. The methanol extract of Temu lawak has activity as an ACE inhibitor with an inhibitory percentage of 71.1% at a concentration of 100 ppm²⁵. Temu lawak also contain curcumin and has antioxidant activity³¹. Compounds that act as antioxidants are water-soluble phenolic compounds, namely xanthorrizol, flavonoids, and benzophenones, primary antioxidants³². Tinospora crispa/ Brotowali has pharmacological effects, such as analgesics, antipyretics, lowering blood sugar levels, and antihypertensives. Brotowali contains chemical compounds including alkaloids, soft resin, starch, glycosides, picroretosid, harsa, bitter substances picroretin, tinocrisposid, berberine, palmatin, columbine, and kaokulin or picrotoxin. The mechanism of Tinospora's antidiabetic effect is by inhibiting the alpha-glucosidase enzyme, which plays a role in the conversion of carbohydrates to glucose. The main component that acts as an antidiabetic is terpenoids³³. Orthosiphon stannous (Cat's whiskers) has various pharmacological effects, namely to treat rheumatism, fever, hepatitis, kidney stone laxative, hypertension, diabetes, and epilepsy. Cat's whiskers contain chemical compounds, including steroids, oleanolic acid, polyphenols, flavonoids, and terpenoids. The polyphenol compounds in cat whiskers have an antioxidant effect by preventing the formation of lipid peroxides and preventing oxidative stress. The antihypertensive effect of cat's whiskers through a diuresis mechanism by increasing sodium excretion in the kidneys. The antioxidant activity of polyphenol compounds in cat whiskers contributes to its antihypertensive effect. The antidiabetic effect of cat whiskers' water extract through the mechanism of increasing body metabolism and glucose utilization to energy^34-35. Phyllanthus niruri (meniran) has an antihypertensive mechanism as an ACE inhibitor. Giving meniran leaf aqueous extract to rabbits reduced systolic, diastolic, and mean arterial pressure. Meniran decreases aldosterone production, which plays a role in causing severe hypertension³⁶. The hypotensive effect of aqueous extract of meniran leaves through the mechanism of decreasing myocardial contractility and relaxation of vascular smooth muscle. leaves also have an antidiabetic effect by inhibiting glucose absorption and increasing glucose storage. In vitro the extract inhibited the α-amylase and α-glucosidase enzymes with IC₅0 values of 2.15±0.1mg/ mL and 0.2±0.02mg/mL activities, respectively³⁷. Andrographis paniculata/sambiloto contains flavonoids and terpene lactones and glycosides, such as andrographolide, deoxyandrographolide, 11,12-didehydro-14-eoxyandro-grapholide, and neoandrographolide³⁸. The ethanol extract of sambiloto can prevent myocardial ischemia in isopreterenol-induced rats. Isopreterenol is a catecholamine that can increase damage to the heart muscle because isopreterenol can cause oxidative stress. The chemical content of terpenes lactone, andrographolid, and neoandrographolid have a cardioprotective effect because they have antioxidant activity, free radical scavenging, and anti-lipid peroxidation properties³⁹. Apart from having a cardioprotective effect, sambiloto extract also has an antihypertensive effect. The antihypertensive effect arises because sambiloto can relax the smooth muscle of blood vessel walls. Sambiloto extracts and Andrographolid have hypoglycemic effects in-vitro by inhibiting the enzymes α-glucosidase and α-amylase and increase glucose metabolism. Andrographis paniculata, increase glucose utilization in peripheral tissues via an insulin-dependent mechanism^40-41.

Hypertension in diabetic patients can increase both microvascular and macrovascular complications, so it is necessary to properly manage hypertension to minimize complications and inhibit disease progression⁴². The animal and human studies have demonstrated a link between the used Renin-Angiotensin-Aldosterone System (RAAS) and insulin resistance pathogenesis⁴³. Angiotensin II influences glucose metabolism through its consequences on insulin signaling pathways, decreased tissue blood flow, oxidative stress, increased sympathetic activity, and adipogenesis. Enhanced sympathetic nerve activity leads to elevated catecholamines, preceding endothelial dysfunction and raised blood pressure. Inhibition of RAAS with ACE inhibitors or angiotensin receptor blockers may increase glucose metabolism by preventing angiotensin II formation or preventing Angiotensin II receptors⁹. Insulin resistance also increases sympathetic nerve activity, leading to reabsorption of sodium in the kidneys, contributing to hypertension⁴⁴. The research results show that the herbs with the composition of Curcuma xantorrhizae rhizome, Andrographis paniculata herba, Tinospora crispa caulus, Orthosiphon stannous folium, and Phyllanthus niruri folium have ACE inhibitor and alpha-glucosidase inhibitor activity with an IC₅₀ value of 103.75µg/ml, and 49.95µg/ml, respectively.The extract also has antioxidant activity with an IC₅₀ value of 11.4µg/ml. The administration of ACE inhibitors to diabetic patients can increase insulin sensitivity through several mechanisms, namely reducing angiotensin II formation and preventing bradykinin degradation, prolonging its half-life, and causing relaxation of blood vessels endothelial-dependent mechanisms⁴⁵.

CONCLUSION:

Antihypertensive jamu with the composition of Curcuma xantorrhizae rhizome, Andrographis paniculata herba, Tinospora crispa caulus, Orthosiphon stannous folium, and Phyllanthus niruri folium meet specific and non-specific parameter standards and have ACE inhibitor, alpha-glucosidase inhibitor, and antioxidant activity with an IC₅₀ value of 103.75µg/ml, 9.95µg/ml, and 11.4µg/ml, respectively.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors express their gratitude to the Ministry of Education and Culture and Kemala Bangsa foundation who has funded this research.

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Received on 13.08.2021 Modified on 19.10.2021

Research J. Pharm. and Tech 2022; 15(9):4212-4217.

DOI: 10.52711/0974-360X.2022.00708

Parameter
Water content (%v/w)	Ash content (%w/w)	Ash insoluble acid content	level of substances dissolved in alcohol (%)	Level of substances dissolved water (%)	Coliform microbial contamination (colony/g)	mold / yeast numbers (colony/g)
5.85±0.24	5.92±0.18	0.41±0.07	60,16±1.33	31.93±1.72	negative	negative