TOXICITY TESTING OF JAMU FORMULA FOR ASTHMA Uji Toksisitas Formula Jamu Untuk Asma

TOXICITY TESTING OF JAMU FORMULA FOR ASTHMA

  

Uji Toksisitas Formula Jamu Untuk Asma

Galuh Ratnawati and Zuraida Zulkarnain

  

Medicinal Plant and Traditional Medicine Research and Development Centre

Email: galuhratnagaluh@gmail.com

ABSTRAK

  

Asma adalah gangguan pernapasan yang mempersempit saluran udara karena hiperaktivitas terhadap rangsan-

gan tertentu, yang menyebabkan peradangan; penyempitan ini bersifat sementara. Meningkatnya jumlah orang

yang tertarik dalam penggunaan pengobatan dengan menggunakan tanaman obat dan herbal. Tanaman Obat dan

Penelitian Obat Tradisional dan Pusat Pengembangan (B2P2TO2T) telah mengembangkan rumus jamu yang ter-

diri dari sembung (Blumea balsamifera L. DC.), kemukus (Piper cubeba L.), patikan kebo (Euphorbia hirta L.), dan

rumput teki (Cyperus rotundus L.) untuk pengobatan asma. Sembung secara empiris digunakan sebagai obat asma.

Kemukus yang berisi dihidrokubebin memiliki efek melonggarkan pernafasan. Patikan kebo digunakan untuk men-

gobati asma, gangguan bronkial kronis, radang selaput lendir hidung hidung akut, dan sebagai ekspektoran. Rum-

put teki digunakan sebagai anti-inflamasi. Penelitian ini dilakukan untuk mengetahui toksisitas herbal yang terdiri

sembung, kemukus, patikan kebo, dan rumput teki pada tikus Wistar. Uji toksisitas akut menggunakan 3 kelompok;

kelompok kontrol, kelompok dosis 5.000 dan 2.500 mg/kg bb. Sedangkan uji toksisitas subkronis menggunakan

empat kelompok; kelompok kontrol, dosis 0,144; 0,288; 0,576 g /200 g bb. Data dikumpulkan untuk gejala toksik

dan kematian, berat badan, kimia darah dan pemeriksaan histologis dari organ-organ vital. Hasil uji toksisitas akut

menunjukkan hingga dosis maksimum 5.000 mg/kg bb menunjukkan tidak toksik. Uji toksisitas subkronis, histologi

menunjukkan organ vital tidak menunjukkan perubahan tertentu dalam kedua kelompok kontrol dan dosis kelom-

pok.

  Kata kunci: toksisitas,Blumea balsamifera L. DC., Piper cubeba L., Euphorbia hirta L.,and Cyperus rotundus L.

  

ABSTRAC

Asthma is a respiratory disorder that narrows the airway because of hyperactivity to certain stimuli, which cause

by inflammation; this narrowing is temporary. Increasing number of people are interested in the use of treatment

using medicinal plants and herbs. Medicinal Plant and Traditional Medicine Research and Development Centre

(B2P2TO2T) has develop jamu formula consisting of blumeae folium (Blumea balsamifera L. DC.), cubebs (Piper

cubeba L.), asthma weed (Euphorbia hirta L.), and nutgrass (Cyperus rotundus L.) for the treatment of asthma.

  

Blumeae folium empirically was used as an asthma medication. Cubebs which contained dihydrocubebin has the

effect of tracheospasmolitic. Asthma weed used to treat asthma, chronic bronchial disorders, acute nasal catarrh,

and as an expectorant. Nutgrass are used as anti-inflammatory. The study was conducted to determine the toxic-

ity of herbs consisting blumeae folium, cubebs, asthma weed, and nutgrass in Wistar rats. Acute toxicity test using

the 3 groups; control group, dose groups 5.000 and 2.500 mg/ kg bw. While the subchronic toxicity test using

four groups; control group, a dose of 0.144; 0.288; 0.576 g/200 g bw. Data were collected for toxic symptoms and

mortality, body weight, blood chemistry and histological examination of vital organs. Acute toxicity test results

show up to a maximum dose of 5.000 mg/kg bw showed practically not toxic. Subchronic toxicity test, histology

showed the important organs showed no specific changes in both the control group and dose groups.

  Keywords: toxicity, Blumea balsamifera L. DC., Piper cubeba L., Euphorbia hirta L.,and Cyperus rotundus L.

  INTRODUCTION

  Asthma including five major causes of death in the world, reaching 17.4 percent. While in Indonesia, the disease is among the top ten causes of morbidity and mortality. The World Health Organization (WHO) noted that there are currently 300 million asthma suffer- ers worldwide. Indonesia alone has 12.5 mil- lion people with asma (Anonim, 2008; Sarbini, 2009). Asthma is a respiratory disorder that is narrowed because of hyperactivity to certain stimuli, which cause inflammation, this condi- tion is temporary. The increasing number of people who are interested in the use of treat- ment using herbs and spices makes a research and development regarding the efficacy and safety of herbal medicinal plants becoming very important. Blumeae folium, cubebs , asth- ma weed, and nutgrass are some medicinal plants that have been used for the treatment of asthma individually. Combination using the four medicinal plants are being developed with the hope of synergism interaction increased the efficacy as antiasthma. Scientific evidence about the safety of this herbs is absolutely nec- essary.

  Blumeae folium (Blumea balsamifera L. DC.) contains flavonoids, borneol, cineol, limonene, camphor, a-pinene, Camphene, b- pinene, 3-Carene, sesquiterpenes, monoter- penes, triterpene, and cryptomeridiol, blumea- lactone A, B, C. Blumeae folium empirically used in the treatment of respiratory diseases such as sinusitis, colds, asthma and bronchitis as an expectorant (Globinmed, 2014).

  Cubebs (Piper cubeba L.) is character- ized by lignan cubebin, essential oil com- pounds such as cadinol, a tricyclic sequiter- pene, alcohol, a mixture of isomer cardinenes, 1,4-cineol and terpineol-4 (Wagner, 1996). Di- hydrocubebin have tracheospasmolytic effect, reducing the effect of the contraction of meta- kolin (Wahyono dkk., 2003).

  Asthma Weed (Euphorbia hirta L.) con- tains quersetin, quercitrin, xanthorhamnin, leucocyanidin, 3,5-diglucoside, tanning sub- stances with gallic acid as a constituent com- ponent, inocyte, sterols, terpenoids such as taraxerol, friedelin, n-alkanes, and triacontane.

  Euphorbia hirta var. procumbens

  (Boiss) Small- containing herfirdelin, β-amarin, β-cytosterin, hentriakontan (BPOM, 2006). This herb used for the treatment of lung abscess, chronic bronchitis, asthma, dysentery, inflammation of the mammary gland or breast and typhoid ab- dominalis (Hariana, 2006).

  Nutgrass (Cyperus rotundus L.) contains alkaloids, flavonoids, tannins, starch, glyco- sides, furochromones, monoterpenes, sesqui- terpenes, cytosterol, fatty oil containing waxy substance that is neutral, glycerol, linoleic acid, myristic and stearic acid (Singh et al., 2012). The aim of this study was to make sure the safety of anti asthma herbs formula.

  MATERIALS AND METHOD Materials

  This research was exploratory experi- mental design (CRD) with one factor infusion dose variation. The study was conducted in the Animal Laboratory, Medicinal Plant & Tradi- tional Medicine R&D Center, Tawangmangu in April-December 2012. Fourty two male and fe- male Wistar rats, age 2-3 months with a body weight of 180-230 g. Mice were obtained from the Animal House Center for R & D TO & OT Tawangmangu. Determination of the number of samples used the formula: T (n-1)> 15. “T” was the number of groups, and “n” was the number of samples in each group.

  The tools used for acute and subchronic toxicity tests include pots infusion, stomach probes, syringes, microhematocrit, centrifuge tube, centrifuge equipment, and minor sets. ^{TOXICITY TESTING OF JAMU FORMULA FOR ASTHMA Uji Toksisitas Formula Jamu Untuk Asma}

  Methods Acute Toxicity Test

  Herbs consists of blumeae folium, cubebs, asthma weed, and nutgrass, with a ra- tio of 5: 3: 3: 5. Eighteen Wistar rats males and females were randomly divided into 3 groups, namely: Control group was given distilled water Group 1: given a dose of herb 5.000 mg / kg bw.

  Group 2: given a dose of herb 2.500 mg/ kg bw.

  Each rats were weighed to determine the volume of a given infusion. Infusion was given on the first day and then was observed 1-4 hours after infusion of the presence of symp- toms of vomiting, diarrhea, tremors, seizures, losing appetite, weakness, difficulty breathing, etc. Further observations were made every day for 14 days. Every two days weighing is done to determine the effect of body weight on the test material body weight gain. If found death necropsy performed, then made observation to vital organs, such as liver, kidney, heart, spleen, lungs and stomach both macroscopic and microscopic. Histopathological prepara- tions stained with hematoxylin eosin.

  Subchronic Toxicity Test

  Twenty four male and females Wistar rats were randomly divided into 4 groups, namely: Control group was given distilled water Group 1:herbs infusion dose 0.144 g/200g bw Group 2:herbs infusion dose 0.288 g/200g bw Group 3:herbs infusion dose 0.576 g/200g bw

  Each rats were weighed to determine the volume of a given infusion. Infusion was given for 90 consecutive days. Observations were made on the presence of symptoms of vomiting, diarrhea, tremors, seizures, losing appetite, weakness, difficulty breathing, etc..

  Further observations were made every day for 90 days.

  Each body weight weighing is done once a week to determine the effect of test materi- als on body weight gain. Blood sampling was performed on day 0, 45th, and 90th for blood chemistry values include urea, creatinine, SGPT, SGOT. If found death necropsy performed, then made observation to vital organs, such as liver, kidney, heart, spleen, lungs and stomach both macroscopic and microscopic. Histopatho- logical preparations stained with hematoxy- lin eosin. At the end of the study all animals sacrificed and then necropsy performed, then made observation to vital organs, such as liver, kidney, heart, spleen, lungs and stomach both macroscopic and microscopic. Histopathologi- cal preparations stained with hematoxylin eo- sin.

  Data were analyzed using one-way ANO- VA test.

  Acute toxicity test results showed no significant difference in body weight between the dose groups and the control group (Ta- ble 1). No symptoms of vomiting, diarrhea, tgemetar, seizures, not eating, lethargy, short- ness of breath found on all test animals. There was no death during the period of testing and observation.

  Galuh Ratnawati and Zuraida Zulkarnain

RESULTS AND DISCUSSION

TOXICITY TESTING OF JAMU FORMULA FOR ASTHMA

  _{Uji Toksisitas Formula Jamu Untuk Asma}

Table 1. Rats Body Weight at Acute Toxicity Test

  Body weight (g) Day 0 Day 6 Day 14 Bw Gained Male Control

  264.33±3.21 261.67±2.89 313.33±4.16 49.00±5.57 251.67±41.43 242.67±9.61 286.00±8.54 34.33±4.62 Dose 2500 mg/kg bw

  249.33±19.50 246.67±23.29 292.67±21.39 43.33±2.52 Dose 5000 mg/kg bw Female Control

  187.33±17.21 190.00±14.18 207.67±13.58 20.33±4.73 172.00±12.77 179.00±16.09 195.00±12.49 23.00±1.00 Dose 2500 mg/kg bw

  182.67±12.42 184.00±10.15 205.67±10.41 23.00±9.54 Dose 5000 mg/kg bw Data was Mean ± SD

• Significantly different compared to control, p<0.05

  Subchronic toxicity test results showed no significant difference in body weight be- tween the dose groups and the control group (Table 2). No symptoms of vomiting, diarrhea, trembling, seizures, not eating, lethargy, short- ness of breath found on all test animals. There was no death during the period of testing and observation.

  Examination of renal function param- eters were the levels of urea and creatinine in the blood. The results showed significantly difference in urea levels in female animals be- tween dose group 0.576 g/200 g bw with the control group on day 45 and on day 90, and still in the normal range. Creatinine levels of males dose groups 0.144 and 0.288 g/200 g bw with the control group at day 45, were still in the normal range. Similarly, the creatinine levels on day 90 male dose groups 0.288 and 0.576 g/200 g bw.

  

Table 2. Rats Body Weight at Subchronic Toxicity Test

Body weight (g)

Day 0 Day 30 Day 60 Day 90 Bw gained

  Male Control 247.00±36.17 332.67±55.15 380.33±54.98 400.00±52.05 153.00±16.52

  200 g bw 228.33±16.17 317.33±22.03 367.00±33.78 385.67±28.29 157.33±13.58 Dose 0.144 g/

  222.67±8.50 325.00±9.54 344.00±6.93 366.33±4.93 143.67±10.12 Dose 0.288 g/200 g bw

  236.33±18.77 335.67±25.38 364.33±38.89 381.33±33.50 145.00±19.08 Dose 0.576 g/200 g bw Female Control

  198.67±3.79 230.00±6.00 248.67±3.21 252.67±1.53 54.00±5.29 184.00±3.61 218.33±7.57 236.33±10.02 241.67±1.53 57.67±3.06 Dose 0.144 g/200 g bw

  173.33±8.96 220.33±23.07 232.33±28.11* 241.33±33.53 68.00±25.63 Dose 0.288 g/200 g bw

  172.00±14.11 209.00±18.73 217.33±24.85* 224.33±23.97* 52.33±12.50 Dose 0.576 g/200 g bw Data was Mean ± SD

• Significantly different compared to control, p<0.05

  Galuh Ratnawati and Zuraida Zulkarnain _{Satellite 66.33±6.11 35.33±2.08 39.33±1.53 1.17±0.12 0.70±0.00 0.47±0.06 Male Day 0 Day 45 Day 90 Day 0 Day 45 Day 90 Urea Creatinin}

Table 3. Chemical Component in Serum

Dose 0.144 g/ 200 g bw 64.67±19.76 48.33±6.11 50.00±1.73 1.40±0.00 0.80±0.17* 0.43±0.06 Dose 0.288 g/ 200 g bw 48.67±4.73 39.00±7.55 51.67±2.08 1.27±0.15 0.90±0.36* 0.40±0.00* _Female Dose 0.576 g/ 200 g bw 58.67±14.22 37.00±7.21 46.33±7.02 1.07±0.06 0.73±0.25 0.50±0.00* Satellite 44.33±13.28 38.33±5.86 39.67±0.58 0.87±0.12 0 80±0.17 0.53±0.06 Dose 0.144 g/ 200 g bw 39.67±4.51 29.33±2.08 36.67±1.53 0.97±0.15 0.90±0.17 0.47±0.12 Dose 0.288 g/ 200 g bw 43.00±13.45 36.33±2.31 50.00±2.65 1.13±0.06 0.80±0.17 0.43±0.06 Dose 0.576 g/ 200 g bw 40.33±11.59 29.00±0.00* 31.67±2.31* 0.93±0.15 0.63±0.12 0.43±0.12 Data was Mean ± SD Data was Mean ± SD

• Significantly different compared to control, p<0.05

  Examination of liver function param- eters were SGPT and SGOT levels in the blood. The results showed significantly difference in the levels of SGPT at doses of 0.144 g/200, 0.288, 0.576 g/200 g bw group of females with the control group on day 90. However, SGPT levels were higher than the range since before giving jamu formulas for asthma (Day 0).

  The histologic features in vital organs showed no specific changes were observed ei- ther in the control group and group dose. The composition of cells and tissues appear normal as shown in Figure 1.

  

Table 4. Enzyme Activities in Serum

_{Male Day 0 Day 45 Day 90 Day 0 Day 45 Day 90} SGPT SGOT

  

Control 44.67±4.04 35.67±5.03 31.67±7.02 171.00±48.28 143.33±13.01 110.00±16.37

Dose 0.144 g/ 200 g bw 45.33±2.08 39.67±1.53 34.33±7.57 219.00±35.54 147.33±26.27 130.00±49.57

Dose 0.288 g/ 200 g bw 42.67±10.21 51.00±2.00 40.00±7.55 206.67±13.43 112.00±11.53 143.67±31.26

_Female

Dose 0.576 g/ 200 g bw 42.00±4.58 43.33±7.77 39.67±0.58 176.33±26.69 177.00±64.86 122.00±14.73

Control 40.67±8.50 45.00±7.00 59.00±13.53 219.00±20.07 190.67±28.29 178.00±50.27

Dose 0.144 g/ 200 g bw 37.00±1.00* 38.67±0.58 35.33±3.79* 209.67±22.01 226.33±54.42 168.33±62.79

Dose 0.288 g/ 200 g bw 40.00±13.08 42.67±10.97 46.67±12.22* 196.00±7.81 183.33±11.93 183.67±62.39

Dose 0.576 g/ 200 g bw 44.33±0.58* 38.67±1.53 46.33±9.02* 211.00±8.72 207.67±67.34 215.00±72.81

Data was Mean ± SD

• Significantly different compared to control, p<0.05

  Results of acute toxicity tests showed no symptoms of toxicity and mortality in all test animals up to the largest dose given at 5.000 mg/kg bw. Thus classified jamu formu- las for asthma practically not toxic. Liver func- tion tests performed by examination of liver enzymes in the blood, namely SGOT (Serum Glutamic oxaloacetic transaminase) and SGPT (Serum Glutamic Pyruvic Transaminase). In normal conditions the SGOT produced by the liver, cardiac muscle, kidney, brain, and red blood cells. This enzyme is released into the bloodstream when these tissues are damaged. For example, the blood SGOT levels increased in conditions of muscle injury and heart attack. Therefore, the increase of this enzyme in the blood does not mean that the liver tissue dam- age as it can be elevated in conditions other than liver damage. Unlike the SGOT, SGPT is a specific indicator of liver cell damage. This en- zyme is also produced in small amounts by the kidney and striated muscle (Anonim, 2014). SGOT and SGPT describe liver cell damage if the value is greater than or equal to 3 times the normal value.

  Urea and creatinine are the end product of nitrogen metabolism.

  Urea is the primary metabolite derived from protein and tissue protein turnover. Creati- nine is a product of the catabolism of muscle creatine. Both are relatively small molecules (respectively 60 and 113 daltons) which dis- tributes the total water in the body (Wikipe- dia, 2014) . Creatinine levels are within nor- mal limits is 0.2-0.8 mg/dl (Mitruka, 1981).

  Renal conditions can be described by the ra- tio of urea and creatinine. Urea and creatinine ratio> 100:1 indicates pre-renal damage, 40- 100: 1 indicates normal or post-renal damage, <40:1 indicates intrarenal damage (Wikipedia, 2014). The ratio of serum urea and creatinine in subchronic toxicity tests is 92.66: 1 for male and 73.65: 1 for female at the highest dose, which means that the kidneys in normal con- ditions.

  CONCLUSION

  Jamu formulas for asthma is practically not toxic and didn’t damage liver also kidney.

  ACKNOWLEDGEMENT

  Thank to Traditional Medicine and Me- dicinal Plant Research and Development Cen- ter, Health Ministry of Indonesia for support- ing fund through DIPA 2012.

  Figure 1. Histology of organs in the control group (left) and dose group (right) with a magnification of 200x, 100x except stomach. No specific changes in

both groups (a. heart, b. lung, c. stomach, d. liver, e. spleen, f. kidney) ^{TOXICITY TESTING OF JAMU FORMULA FOR ASTHMA Uji Toksisitas Formula Jamu Untuk Asma} Galuh Ratnawati and Zuraida Zulkarnain Analysis, A Thin Layer Chromatography

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