Orthosiphon stamineus : Ethnopharmacology, Extraction &

Bioprocesses and Anti-Cancer activities

(Paper Review)

Submitted By

Qabas Marwan Abdulazeez

Metric No: G1223171

M. Sc. Student

Department of Biotechnology Engineering

Date of Submission: 15th May 2014

Submitted To

Assoc. Prof. Azura BT. Amid

Department of Biotechnology Engineering

ABSTRACT

Ethnopharmacological relevance: Orthosiphon stamineus is a medicinal herb belong to

the family Lamiaceae. It is a widely used in several countries (especially in Indonesia and

Malaysia) as traditional medicine. It was known as Java tea or Misai Kuching. This

plant has several activities, such as antioxidant, antibacterial, diuretic and anticancer.

Because of those properties, O. stamineus is potential to be developed as a new source of

drugs and to treat some critical human diseases

Aim of the review: The present paper reviewed the ethnopharmacology, extraction &

bioprocesses anti-cancer activities.

Materials and methods: Information on O. stamineus was gathered via the Internet using

Google Scholar and libraries. Additionally, information also was obtained from some

electronic books. The keywords applied on Google was Orthosiphon stamineus, java tea,

Misai Kuching, O. stamineus extraction, O. stamineus anti-cancer.

Results: More than 116 chemical compounds have been isolated, and the main

compositions are essential oils, organic acids and flavones, etc. Orthosiphon stamineus

and its active principles possess wide pharmacological actions, such as anti-cancer,

antibacterial, anti-oxidative and Diuretic activities.

Conclusions: As an important traditional Malaysian and Indonesian medicine, further

studies on O. stamineus can lead to the development of new drugs and therapeutics for

various diseases, and how to utilize it better should be paid more attentions.

CONTENTS

1. Introduction 1

2. Ethnopharmacology Studies and Activities 3

2.1 Anti-oxidant Activity 3

2.2 Diuretic Activity 5

2.3 Anti-cancer Activity 5

2.4 Anti-diabetic 7

2.5 Anti-Fungal Agents 8

2.6 Other Activities 9

3. Extraction & Bioprocesses 10

4. Chemical Compounds 10

5. Conclusion 15

6. References 16

1

1. Introduction

Orthosiphon stamineus plant is a popular medicinal plant in Southeast Asia. It is

widely used for treatment of kidney, bladder inflammation, gout and diabetes (Akowuah,

Ismail, Norhayati, & Sadikun, 2005), especially those affecting the urinary tract, diabetes

mellitus, hypertension, rheumatism, tonsillitis and menstrual disorder (Awale, et al.,

2003). Several compounds have been isolated from this plant which includes sinensetin,

an important flavonoid having an antioxidation property. It has high chemo-sensitizing

effect which is used to synthesize multi-drug resistance (MDR) cell for anticancer drugs

(Ahmad, et al., 2008).

Although it was first introduced to the European consumers as an herbal tea in the

20th century, this species was only became popular, particularly in Indonesia and

Malaysia in the last few decades. In Malaysia, it has been used in traditional medicine to

treat diseases of the urinary system (Adam, et al., 2009), and used as diuretic, and to treat

rheumatism, abdominal pain, kidney and bladder inflammation, edema, gout and

hypertension (Abdul Majid, 2010). It is believed that the bioactive compounds contained

in the leaves of O. stamineus exert the effects as an antiallergic, antihypertensive, anti-

inflammatory, antioxidant , and diuretic properties (Abdullah, Shaari, & Azimi, 2012).

Orthosphon stamineus is a perennial herb. It attains 0.3-1.5 m high and having 4-

angle stem. Leaves are simple, opposite, ovate-oblong-lanceolate, elliptic or rhamboid,

which have 2-4 cm wide and 4-7 cm long. The flowers are white, blue or violet. When

the flowers open, stamens and pistil extend out far beyond the petals, which create "cat's

whiskers" effect. This plant needs full sun and a moist garden soil for growing and

2

flowering. However, it grows perfectly under light shade and warmer climates (Adnyana

, Setiawan , & Insanu, 2013) . O. stamineus has many other names, include: Orthosiphon

aristatus, Orthosiphon spicatus, Orthosiphon blaetter, kutum, mamam, bunga laba-laba,

remuk jung, remujung, kumis ucing, songot koceng, sesalaseyan (Java), java tea, cats

whisker, Indian kidney tea (English), mao xu cao (China), misai kucing, ruku hutan

(malaysia), kabling gubat, kabling parang (Philippine), se-cho, myit-shwe (Myanmar),

rau-meo (Vietnam), neko no hige (Japan), katzenbart (Germany) and yaa-nuad-maew, pa-

yab-mek (Thailand). (Abdul Majid, 2010) (Anonymous, 1967).

Figure (1): Orthosiphon stamineus (Misai kucing).

Source : http://bestfarms.com.my/wp-content/uploads/2011/07/bunga_misai_kucing.jpg

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2. Ethnopharmacology Studies and Activities

O. Stamineus has been traditionally used for many diseases, especially in South

East Asian countries like: Indonesia, Malaysia, Thailand, Vietnam and Myanmar. It is

also recognized in European country, such as Holland, France and England to be

consumed as a herbal product. The leaves of this plant have been used in Indonesia for

diuretic, preventing and treating rheumatism, diabetes mellitus, hypertension, tonsillitis,

epilepsy, menstrual disorder, gonorrhoea, syphilis, renal calculi, gallstone, acute and

chronic nephritis, gout arthritis, and antipyretic.

Many of the beneficial effects of O. Stamineus consumption have been attributed

to the pharmacological actions of these major constituents (Pan, et al., 2011). In Vietnam,

the aerial part is used for treating urinary lithiasis, edema, eruptive fever, influenza,

hepatitis, jaundice and biliary lithiasis (Anonymous, 1967), while in Malaysia and

Thailand, the leaves are used for diuretic, kidney tonic, and cystotonic medicines

(Adnyana , Setiawan , & Insanu, 2013).

Many Ethnopharmacological Activities for O. Stamineus have been discovered

by many studies, the most important activities are :

2.1 Anti-oxidant Activity

Recently, there has been considerably interest in finding naturally occurring

antioxidants to replace synthetic antioxidants in foods and medicinals. Several studies

have analyzed the antioxidant potential of a variety of herbs. Among the different parts of

plants studied, the leaves of O. stamineus are reported to have extracts with free radical-

scavenging activity (Akowuah, Zhari, Norhayati, Sadikun, & Khamsah, 2004) .

4

The highest antioxidant activity was found in acetone extract. Other report

observed that there were variations in total phenolic compounds, ranging from 6.7 to 10.1

mg caffeic acid/g dry weight of the methanol extract. Some studies also proved using

different in vitro method (superoxide scavenging and xanthine oxidase) that O. stamineus

extract showed potential antioxidant activity (Adnyana , Setiawan , & Insanu, 2013).

(Akowuah, Zhari, Norhayati, Sadikun, & Khamsah, 2004) study reported that

until now, Twenty phenolic compounds were isolated, including nine lipophilic flavones,

two flavonol glycosides, and nine caffeic acid derivatives, such as rosmarinic acid and

2,3- dicaffeoyltartaric acid, were identified and quantified by HPLC analysis.

More than fifty compounds were isolated from O. Stamineus. They were tested

their antioxidant activity using inhibition of NO production in LPS-activated

machropage-like J774.1 cells. Siphonol A C and E, 2-O-deacethylorthosiphol J,

orthosiphol A, B, D, H, K, M, N, O, X, Y staminol A, neoorthosiphol B, Staminols C and

D, orthosiphonones C and D and 14-deoxo-14-O-acetylorthosiphol Y,

nororthosiphonolide A, orthosiphonone A, secoorthosiphols B and C and 3-O-

deacetylorthosiphol I showed stronger antioxidative activity than NG-monomethyl-L-

arginine as positive control. 2-O-deacetylorthosiphonone I, showed the most potent

activity, with an IC50 value of 35.0 M. (Adnyana , Setiawan , & Insanu, 2013). The

plants strong anti-oxidant action is what makes many researchers to explore the potential

pharmacological properties of this plant (Abdul Majid, 2010).

5

2.2 Diuretic Activity

One of earlier research works reported that, because of the high content of

potassium, inositol and lipophilic flavones in the leaves of O. stamineus it possesses

strong diuretic activity. (Abdul Majid, 2010). Diuretic activity of hydroalcohol extract

from aerial parts of O. stamineus was reported. At a dose of 50 mg/kg, this extract

showed similar effect with hydrochlorotiazide at a dose of 10 mg/kg (Beaux, Fleurentin,

& Mortier, 1999). Other studies reported that an aqueous extract and leaves tincture

enhanced ion excretion of rats which were not due to the potassium content of the starting

material (Englert & Harnischfeger, 1992) (Cicero, Sando, Izzo, Vasta, Trimarco, &

Borghi, 2012)..

A study for (Kannappan, Madhukar, Marymmal, Sindhura, & Mannavalan, 2012)

showed that the administration of methanol extract at doses of 100 and 200 mg/kg bw

significantly decreased serum creatinine levels, blood urea, urinary protein and extent

renal damage after 10 days administration.

The results of (Arafat, Tham, Sadikun, Zhari, Haughton, & Asmawi, 2008) study

showed that, sodium and potassium excretion increased significantly (p < 0.05 and

6

2012). Sinensetin has a wide range of biological activities such as antibacterial,

antifungal, antitumor, anticancer, prostaglandin binding and insect antifeedant (Hossain

& Ismail, 2012).

Many studies investigated about the Sinensetin in O. stamineus and the

Anticancer Activity, especially for breast cancer. Over 60% of breast cancer cases are

Estrogen Receptor (ER+) positive which is highly dependent on estrogen for growth.

Tamoxifen is a potent antagonist of ER and has been approved to be the first line anti-

estrogen therapy since the early 1970s . However, most tamoxifen sensitive breast cancer

cases succumb to tamoxifen resistance leading to poor prognosis . The resistance that is

developed is mainly due to cross-talk between ER and type 1 tyrosine kinase receptors,

including Epidermal Growth Factor Receptor (EGFR) and human epidermal growth

factor receptor 2 (HER2). These two cytokines are forund to be up-regulated during

tamoxifen treatment leading to further progression of breast cancer. In addition, up-

regulation of bcl-2 and cycloxigenase-2 (COX-2), which are key players in tenor

development, also contribute to tamoxifen resistance (Sahib, Ismail, Othman, & Abdul

Majid, 2009). O. stamineus as Synergistic enhancer to tamoxifen, it may probably exert

the anti-estogenic effect (Abdul Majid, 2010). In (Dolekov, Rrov, Grz, Vondrusov,

Strnad, & Krytof, 2012) study, the antiproliferative properties of a chloroform extract of

the leaves of O.stamineus and of pure eupatorin. The compound was able to reduce the

number of viable cancer cells to the same extent as the extract, with IC50 values in

micromolar range.

Recently, methanolic extract of O. stamineus was found to have potent anti-

angiogenic activity in vitro. However, molecular modeling data and immune-

7

histochemical analysis reveal that this activity occurs due to direct inhibition of the

Vascular Endothelial Growth Factor Receptors (VEGFR). Previous studies have shown

that (VEGFR) are present on the surface of hormone sensitive breast cancer cell line

MCF-7. (Sahib, Ismail, Othman, & Abdul Majid, 2009).

2.4 Anti-diabetic

In (Sriplang, Adisakwattana, Rungsipipat, & Yibchok-anun, 2007) study, when

oral glucose tolerance is tested, the water extract at doses of 0.21.0 g/kg significantly

decreased plasma glucose concentration in dose-dependent manner for both normal and

diabetic rats. At a dose of 1.0 g/kg showed similar effect with glibenclamide (5 mg/kg).

In diabetic rats, after they were given the extract orally (0.5 g/kg) for 14 days, plasma

glucose concentrations were reduced significantly. In addition, plasma triglyceride

concentration was also lower in the extract-treated diabetic rats than that of untreated

group.

Other study showed antidiabetic effects of the petroleum, chloroform, methanol

and water extracts. Chloroform extract at a dose of 1 g/kg bw significantly reduced blood

glucose level. Further, this extract was fractionated and finally one subfraction showed

similar antidiabetic effect with metformin (Mohamed, Mohamed, Asmawi, Sadikun,

Ebrika, & Yam, 2011).

In (Mohamed, Yam, Ang, Mohamed, & Asmawi, 2013) study, the results showed

that, the chloroform sub-fraction 2 (Cf 2-b) of O.stamineus extract at a dose of 1 g/kg

twice daily on diabetic rats for 14 days showed a significant lowering (p < 0.05) of the

final blood glucose level compared to the pretreatment level.

8

2.5 Anti-Fungal Agents

The increasing social and economic implications caused by fungi means there is a

constant striving to produce safer food crops and to develop new anti-fungal agents. In

general, plant-derived essential oils are considered as non-phytotoxic compounds and

potentially effective against plant pathogenic fungi. In recent years, interests have been

generated in the development of safer anti-fungal agents such as plant-based essential oils

and extracts to control phytopathogens in agriculture. Several publications have

documented the anti-fungal activities of essential oils and plant extracts. (Hossain, Ismail,

Rahman, & Kang, 2008).

In 1985, Gurin and Rveillre tested a hydro-alcoholic extract of Orthosiphon

stamineus (DER = 20%) against 9 fungal species. They showed it inhibited the spore

germination in 6 fungal species (Saccharomyces pastorius, Candida albicans, Rhizopus

nigricans, Penicillium digitatum, Fusarium oxysporum, Trichophyton mentagrophytes)

and delayed the growth of remaining species (Aspergillus fumigatus, Aspergillus niger,

Botrytis cinerea). The authors mentioned that the antifungal activity of Orthosiphon

stamineus had not been established before. Consequently, further research on this plant

had to be performed ((HMPC), 2010). The hydrodistillation of the leaves and stems of O.

stamineus gave dark yellowish oils with the major components of the oils having

oxygenated monoterpenes and sesquiterpenes, and their respective hydrocarbons

(Hossain, Ismail, Rahman, & Kang, 2008). Same study concluded that, the active

antimicrobial compounds of essential oils are terpenes derived from O. stamineus, which

are phenolic in nature, it would seem reasonable that their antimicrobial or anti-fungal

mode of action might be related to that of other compounds.

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2.6 Other Activities

O. stamineus as a remedy for kidney stones and gout Deposition of precipitates of

calcium oxalate crystals lead to the condition commonly known as kidney stones, which

can be easily diagnosed by radiological studies or ultrasound examination. This kind of

crystal is difficult to be dissolved and expelled successfully out from the body with

clinical therapy, however certain medications proved to be act as prophylactic agents in

preventing the calcium stones if they have a propensity to recur (Abdul Majid, 2010).

O. stamineus is listed in the French, Indonesia, Dutch, and Swiss pharmacopoeias

for conditions related to renal cleansing and function, and related disorders that include

nephritis, cystitis, and urethritis. In Europe, people use the leaves of O. stamineus extract

as a tonic for kidney and bladder stones, liver and gallbladder problems and urinary tract

infections. It is also used to reduce cholesterol and blood pressure. Researchers have

found it to be mildly antiseptic as well (Abdul Majid, 2010).

(Ho, Noryati, Sulaiman, & Rosma, 2010) study showed that the highest

concentration of rosmarinic acid had the best antibacterial and free radical scavenging

activities. This suggests that rosmarinic acid content is closely associated with

antibacterial and free radical scavenging activities of O.stamineus extracts.

The anti-hyperglycemic effect of O. stamineus was evaluated by (Sriplang,

Adisakwattana, Rungsipipat, & Yibchok-anun, 2007) study. It found the evidence of a

direct stimulatory effect of the Orthosiphon stamineus extract on insulin secretion from

the pancreatic beta cells.

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3. Extraction & Bioprocesses

Leaf powder of Orthosiphon stamineus was extracted with the following solvents;

distilled water, 50% aqueous methanol, methanol, 70% aqueous acetone and chloroform,

at 2, 4 and 8 h, respectively, on a water bath at 40 C (Akowuah, Ismail, Norhayati, &

Sadikun, 2005). The plant was oven-dried at 40 C and the leaves were separated and

grounded into fine powder. The powdered material (360 g) was extracted sequentially by

adding 30 g in each flask with 200 mL of petroleum ether with continuous shaking for

8h. The residue will be dried and extracted successively with chloroform, methanol and

water. The extracts were concentrated under vacuum at 40 C and freeze dried under

vacuum (Sahib, Ismail, Othman, & Abdul Majid, 2009) (Hossain, Ismail, Rahman, &

Kang, 2008) (Adam, et al., 2009).

In some studies, the extraction process will be different from the others. For

example, in (Hossain & Ismail, 2012) study, the dry powder methanol will heat below 40

C with ultra-sonication for 30 min . (Abdullah, Shaari, & Azimi, 2012) study showed

that Stamineus leaves were dried by using three different methods; an oven at 40 oC,

under the shade and directly under the sun were dried for 4 days.

4. Chemical Compounds

Orthosiphon stamineus extractions were more than hundreds chemical

compounds, these compounds were reported and classified as terpenoids (monoterpenes,

diterpenes, triterpenes), polyphenols (lipophilic flavonoids and phenolic acids), saponins,

flavonoids, polyphenols (lipophilic flavonoids and phenolic acids), organic acids, etc, as

shown in Table (1). (Adnyana , Setiawan , & Insanu, 2013) (Rahman & Hossain, 2011).

11

Table (1): Chemical compounds present in Orthosiphon stamineus Benth

NO Chemical compounds NO Chemical compounds

1 1-octen-3-ol 30 Betulinic acid

2 2,3-dicaffeoyltartaric acid 31 Borneol

3 2-O-deacetylorthosiphol J 32 Bornyl acetate

4 3-hydroxy-5,6,7,4-tetramethoxyflavone 33 Caffeic Acid

5 3-O-deacetylorthosiphol I 34 Caffeic acid depside A

6 4-hydroxy-5,6,7-trimethoxyflavone 35 Caffeic acid depside B

7 4,5,6,7-tetramethoxyflavone 36 Caffeic acid depside C

8 5,6-dihydroxy-7,4-dimethoxyflavone 37 Camphene

9 5,7,4'-Trimethylapigenin 38 Camphor

10 5,6,7,4-tetramethoxyflavone 39 Carotol

11 5,7,3',4'-tetramethylluteolin 40 Carvone

12 5-hydroxy-6,7,3',4-tetramethoxyflavone 41 Cis-Caryophyllene

13 6-hydroxyorthosiphol B 42 Caryophyllene oxide

14 6-hydroxy-5,7,4'-trimethoxyflavone 43 Cichoric acid

15 7,3,4-tri-O-methylluteolin 44 Cisimaritin

16 7-O-deacetylorthosiphol B 45 Eugenol

17 14-deoxo-14-O-acetylorthosiphol Y 46 Eupatorin

18 cadinol 47 Hederagenin

19 -humulene 48 Ladanein

20 -bourbonene 49 Limonene

21 caryophyllene 50 Luteolin

22 -elemene 51 Methylripariochromene A

23 -pinene 52 Neoorthosiphol A

24 sitosterol 53 Neoorthosiphol B

25 carryophylene 54 Neoorthosiphone A

26 -hydroxybetulinic acid 55 Norstaminol A

27 -cadinene 56 Norstaminol B

28 Acetovanillchromene 57 Norstaminol C

29 Aurantiamide acetate 58 Norstaminolactone A

12

59 Nororthosiphonolide A 88 Orthosiphol Z

60 Norstaminone A 89 Orthosiphonone A

61 Oleanolic acid 90 Orthosiphonone B

62 Orthochromene A 91 Orthosiphonone C

63 Orthosiphol A 92 Orthosiphonone D

64 Orthosiphol B 93 Pillion

65 Orthosiphol C 94 Quercetin

66 Orthosiphol D 95 Rosmarinic acid

67 Orthosiphol E 96 Salvigenin

68 Orthosiphol F 97 Secoorthosiphol A

69 Orthosiphol G 98 Secoorthosiphol B

70 Orthosiphol H 99 Secoorthosiphol C

71 Orthosiphol I 100 Sinensetin

72 Orthosiphol J 101 Siphonol A

73 Orthosiphol K 102 Siphonol B

74 Orthosiphol L 103 Siphonol C

75 Orthosiphol M 104 Siphonol D

76 Orthosiphol N 105 Siphonol E

77 Orthosiphol O 106 Staminol A

78 Orthosiphol P 107 Staminol B

79 Orthosiphol Q 108 Staminol C

80 Orthosiphol R 109 Staminol D

81 Orthosiphol S 110 Staminolactone A

82 Orthosiphol T 111 Staminolactone B

83 Orthosiphol U 112 Tetramethylscutellarine

84 Orthosiphol V 113 Trans-ozic acid

85 Orthosiphol W 114 Ursolic acid

86 Orthosiphol X 115 Vomifoliol

87 Orthosiphol Y 116 16-hydroxy betulinic acid

Source : (Adnyana , Setiawan , & Insanu, 2013)

13

Moreover, previous research have detected 69 chemical compounds in the

essential oil of leaves of O. Stamineus. They were:

Table (2): Chemical compounds in the essential oil of leaves of O. Stamineus

1-Octen-3-ol -Bourbene -Pinene

2-Methylnapthalene -Ionone Safranal

3-Octanol 2-Pentenyl furane Naphtalene

6-Trimethyl-2-cyclohexe-1 4-dione Isobornylacetate

8-Cineol 4-Heptenal Isomenthone

Benzaldehyde trans 2-Hexanal Heptenal

Borneol Dodecane Eugenol

Camphor 1-Methylnaphtalene -Muniolene

Carvone Cittonellol -Copaene

cis-2-Octenal Limonene Hexanal

cis-Caryophylene 4-dienal trans-Deca-2

Damascenone Dehydroionone cis-Linalooloxide

Geranylacetane -Terpineol Acetophenone

Hexahydrofamesylacetone Hexan-1-ol Germacrene

Linalool trans-Linalooloxide -Cadipene

Methylchavicol -Pinene,Tridecan -Cymene

Phenylacetaldehyde Caryophyllene oxide Camphene

trans- Anethol Germacrene D -Cyclocitral

trans cis-Octa-3-5-dien-2-one Decanal -Elemene

trans trans-Octa-3-5-dien-2-one 2-Amylfurane Menthone

trans-2-(cis)-6- Nonadienale Methyleugenol -Gubebene

Undecan Bornyl Acetate Perillen

-Carryophyllene -Humulene -Elemene

Source : (Hossain, Ismail, Rahman, & Kang, 2008)

14

Figure (2): Structures of Orthosiphon stamineus active compounds: (A) rosmarinic acid,

(B) sinensetin, and (C) eupatorin . (Pan, et al., 2011) .

15

5. Conclusion

As the above said, Orthosiphon stamineus was used and planted in Southeast

Asia. Recently,O. stamineus is widely used in many countries for treatment of kidney,

bladder inflammation, diabetes mellitus, hypertension, rheumatism, tonsillitis and

menstrual disorder. So, there are more potential utilization and development of O.

stamineus out of Asia, especially out of Malaysia.

Obviously, the chemical components and pharmacology activities of O. stamineus

have been studied, and many active compounds were isolated and identified. Of these,

due to the good antibacterial, anticancer, antioxidant and anti-tumor activities, methanol,

acetone and chloroform were used to extract the compound to characterize the quality of

O. stamineus. The different habitat, medicinal parts, extraction methods and solvents,

fresh flowers and dry flowers would result in the different chemical compositions and the

quality of O. stamineus. In Malaysia, O. stamineus was only used to treat some diseases.

But the results of phytochemical researches indicated that this plant capable to produce

many compounds with many activities, such as : anti-oxidant, diuretic, anti-cancer, anti-

diabetic, anti-fungal and anti-bacterial activities, production of essential oils, flavones,

iridoids and so more. these components can be the active parts to treat diseases.

In a word, phytochemical and pharmacological studies of O. stamineus have

received much interest, more and more extracts and active compounds have been isolated

and proved which has the anti-cancer, antiviral, antibacterial, antioxidant and enhance the

immune response effects, etc. Further research especially on in vitro/in vivo antibacterial

and anticancer effects should have priority.

16

6. References

(HMPC), C. o. (2010, March 11). Assessment report on Orthosiphon stamineus Benth., folium. europe: European Medicines Agency.

Abdul Majid, A. M. (2010, Dec 12). Medicinal Potentials Of Orthosiphon Stamineus. webmedcentral, p. 2.

Abdullah, S., Shaari, A., & Azimi, A. (2012, Apr 7). Effect of Drying Methods on Metabolites Composition of Misai Kucing (Orthosiphon stamineus) Leaves. APCBEE Procedia, pp. 2 ( 2012 ) 178 182.

Adam, Y., Somchit, M., Sulaiman, M., Nasaruddin, A., Zuraini, A., Bustamam, A., et al. (2009, Apr 16). Diuretic properties of Orthosiphon stamineus Benth. Journal of Ethnopharmacology, pp. 124 (2009) 154158.

Adnyana , I., Setiawan , F., & Insanu, M. (2013). From Ethnopharmacology To Clinical Study Of Orthosiphon Stamineus Benth. International Journal of Pharmacy and Pharmaceutical Sciences, pp. Vol 5, Issue 3.

Ahmad, M. N., Mee, M. Y., Chew, C., Islam, A. S., Z. I., Surif, M., et al. (2008, Mar 17). Disposable array sensor strip for quantification of sinensetin in Orthosiphon stamineus Benth samples. Microchim Acta, pp. 163: 113119.

Akowuah, G., Ismail, Z., Norhayati, I., & Sadikun, A. (2005). The effects of different extraction solvents of varying polarities on polyphenols of Orthosiphon stamineus and evaluation of the free radical-scavenging activity. Food Chemistry, pp. 93 (2005) 311317.

Akowuah, G., Zhari, I., Norhayati, I., Sadikun, A., & Khamsah, S. (2004, Jan 8). Sinensetin, eupatorin, 30-hydroxy-5, 6, 7, 40-tetramethoxyflavone and rosmarinic acid contents and antioxidative effect of Orthosiphon stamineus from Malaysia. Food Chemistry, pp. 87 (2004) 559566.

Anonymous. (1967). Burmese indigenous medicinal plants : plants with reputed hypoglicemic action. Burma - Myanmar: Burma medical research institute.

Arafat, O., Tham, S., Sadikun, A., Zhari, I., Haughton, P., & Asmawi, M. (2008). Studies on diuretic and hypouricemic effects of Orthosiphon stamineus methanol extracts in rats. Ethnopharmacology, pp. 118 : 354360.

Awale, S., Tezuka, Y., Banskota, A.H., Adnyana, I.K., et al. (2003). Nitric oxide inhibitory isopimarane-type diterpenes from Orthosiphon stamineus of Indonesia. Journal of Natural Products, pp. 66, 255258.

17

Beaux, D., Fleurentin, J., & Mortier, F. (1999). Effect of extracts of Orthosiphon stamineus Benth. Phytother Res, pp. 13 (3):222-25.

Cicero, A., Sando, V., Izzo, R., Vasta, A., Trimarco, A., & Borghi, C. (2012). Effect of a combined nutraceutical containing Orthosiphon stamineus effect on blood pressure and metabolic syndrome components in hypertensive dyslipidaemic patients: A randomized clinical trial. Complementary Therapies in Clinical Practice, pp. 18 (2012) 190 - 194.

Dolekov, I., Rrov, L., Grz, J., Vondrusov, M., Strnad, M., & Krytof, V. (2012).

Antiproliferative and antiangiogenic effects of flavone eupatorin, an active constituent of chloroform extract of Orthosiphon stamineus leaves. Fitoterapia, pp. 83 : 1000-1007.

Englert, J., & Harnischfeger, G. (1992). Diuretic action of aqueous Orthosiphon extract in rats. Planta Med, pp. 58 (03) :237-38.

Ho, C.-H., Noryati, I., Sulaiman, S.-F., & Rosma, A. (2010). In vitro antibacterial and antioxidant activities of Orthosiphon stamineus Benth, extracts against food-borne bacteria. Food Chemistry, pp. 122 : 11681172.

Hossain, M., & Ismail, Z. (2012, Feb 28). Quantification and enrichment of sinensetin in the leaves of Orthosiphon stamineus. Arabian Journal of Chemistry.

Hossain, M., Ismail, Z., Rahman, A., & Kang, S. (2008). Chemical composition and anti-fungal properties of the essential oils and crude extracts of Orthosiphon stamineus Benth. Industrial Crops and Products, pp. 2 7 ( 2 0 0 8 ) 328334.

Kannappan, N., Madhukar, A., Marymmal, Sindhura, P., & Mannavalan, R. (2012). Evaluation of nephroprotective activity of Orthosiphon stamineus Benth extract using rat model. Int J Pharm Tech Res Service, pp. 2(1): 209-15.

Mohamed, E. A., Yam, M. F., Ang, L. F., Mohamed, A. J., & Asmawi, M. Z. (2013). Antidiabetic Properties and Mechanism of Action of Orthosiphon stamineus Benth Bioactive Sub-fraction in Streptozotocin-induced Diabetic Rats. Acupuncture and Meridian Studies, pp. 6(1): 31-40.

Mohamed, E., Mohamed, A., Asmawi, M., Sadikun, A., Ebrika, O., & Yam, M. (2011). Antihyperglicemic effect of Orthosiphon stamineus Benth leaves extracts and its bioassay guided fractions. Molecules, pp. 16 (3787-3801).

Pan, Y., Abd-Rashid, B. A., Ismail, Z., Ismail, R., Mak, J. W., Pook, P. C., et al. (2011). In vitro effects of active constituents and extracts of Orthosiphon stamineus on the activities of three major human cDNA-expressed cytochrome P450 enzymes. Chemico-Biological Interactions, pp. 190 (2011) 1 - 8.

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Rahman, S. M., & Hossain, M. (2011). Isolation and characterisation of flavonoids from the leaves of medicinal plant Orthosiphon stamineus. Arabian Journal of Chemistry, p. 10.1016 (2011) 06.016.

Sahib, H., Ismail, Z., Othman, N., & Abdul Majid, A. (2009). Orthosiphon stamineus Benth. Methanolic Extract Enhances the Anti-Proliferative Effects of Tamoxifen on Human Hormone Dependent Breast Cancer. International Jomml of Pharmacology, pp. 5 (4): 273-276.

Sriplang, K., Adisakwattana, S., Rungsipipat, A., & Yibchok-anun, S. (2007). Effects of Orthosiphon stamineus aqueous extract on plasma glucose concentration and lipid profile in normal and streptozotocin-induced diabetic rats. Journal of Ethnopharmacology, pp. 510514.

Wei , G., Sheen , J., Lu , W., Hwang , L., Ho , C., & CI, L. (2013). Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. Kainan University, Taoyuan: Department of Nutrition and Health Sciences.