CHEMICAL CONSTITUENTS

III. CHEMICAL CONSTITUENTS

A number of studies have been conducted to elucidate the chemical constit- uents and pharmacological activities of the aerial parts of Epimedium herb. Several methods such as column chromatography, liquid chromatography, pulse polarography, coulometric titration, and fluorometry have been used to study the flavonoids from Epimedium species (8). The main constituents in Epimedium plants are the prenylflavone glycosides. Among the important active constituents isolated from the aerial parts of these herbs are the flavonol glycosides (such as icariin and baohuoside I, Fig. 1) and flavonoids.

The glycoside icariin was first isolated from E. macranthum (9). The isolation of icariin from E. brevicornum (10–12), E. koreanum (11,12), E. acuminatum (13,14), E. fargesii (15), E. wushanense (16), E. hunanense (2), and

E. sagittatum (3) was reported. The presence of icariin in Epimedium herb can

be detected by thin-layer chromatography. However, content of other fla- vones might vary in different species (17). Other compounds isolated from Epimedium herb include phenooxy- chromones, flavanoids, chrysoeriol, quercetin, apigenin, apigenin 7,4V-di- methyl ether, kempferol, tricin, luteolin, thalictoside, and brevicornin (8). Luteolin, a flavone, was found to have estrogenic activity with a relative po- tency of 58% compared to genistein (18). Apigenin and quercetin are also estrogenic. Other flavanoids and flavanol glycosides described in Epimedium include epimedokoreanoside I, icariside I, icaritin, epimedoside A, epimedins

232 Yap and Yong

F IGURE 1 Chemical structures of bioactive compounds isolated from Epime- dium: (a) icariin and (b) baohuoside I.

A, B, and C, h-anhydroicaritin, tricin, korepimedoside A and B, sagittato- sides A, B, and C, sagittatins A and B, diphylloside A and B, baohuosides I–VII, and baohuosu (16,19–21). Some of these flavonol glycosides, e.g., baohuoside I (2), were reported to have immunomodulatory activity. The herb also contains ceryl alcohol linolenic acid, oleic acid, palmitic acid, sterols, benzene, tannins, fats, saponins, vitamin E, zinc, and some essential oils.

The quantitative changes of flavonoids in E. koreanum in different col- lecting periods were determined by HPLC and UV spectrophotometry. The result shows that the highest content of flavonoids is found in the flowering period (May) (22).

The seasonal fluctuation of flavonol glycosides in the leaves of E. gran- diflorum var. thunbergianum, E. cremeum, and E. sempervirens (Berberida- ceae) was also investigated (23). The total content of glycosides was greatest at flowering time, and as the leaves mature it became less fluctuating with

a little decrease. The ideal period for the harvest of Epimedium leaves was 2 or

3 months after flowering. By means of RP-HPLC, nine major flavonoids in different parts of five Epimedium plants listed in the Chinese Pharmacopoeia were analyzed and the total contents of nine flavonoids in the four species were found to be highest in the rhizome and roots, followed by leaves and stems (24). The composition of the main constituents and relative contents in the five species were similar in leaves and stems, but different from the rhizome and roots. These differences may affect their pharmaceutical properties.

Epimedium Species 233

IV. SCIENTIFIC BASIS OF THEIR BIOLOGICAL ACTIONS Much of the work on Epimedium has focused on either the whole herb or its

glycosides. Polysaccharide content of extracts may also affect its properties. It should be noted that the vast majority of these studies were performed in mainland China and they have not been replicated elsewhere.

A. Immune Effects Epimedium has been reported to significantly enhance the phagocytic activity

of macrophages (25), to stimulate the migration of T-lymphocytes from the thymus to peripheral organs, and to promote both the proliferation of bone marrow cells and the synthesis of DNA. The n-butanol fraction of E. hunanense and epimedin C isolated from the fraction significantly enhanced the response of spleen antibody-forming cells to nearly normal in the mice treated with the immunosuppressant hydrocortisone acetate. They also markedly enhanced lymphocyte proliferation and caused a significant recov- ery of interleukin-2 (IL-2) production in HCA-treated mice (2). On the other hand, a flavone analog, baohuoside I, isolated from E. davidii was described to significantly prolong mouse heart allograft survival (26). Assessment of the mice for appearance, behavior, biochemistry, hematology, and histology revealed no side effects even at the intraperitoneal dose of 32 mg/kg/day for

14 days. In human studies, Epimedium herbs may regulate immunodeficiency states and relieve the neuroendocrinoimmunological effect inhibited by exogenous glucocorticoid. Studies of Epimedium herbs and the effects on the immune system are summarized in Table 1.

B. Effects on the Skeletal System The effect of Epimedium herb on osteoclastic bone resorption and osteopo-

rosis has been studied in vitro and in vivo using ovariectomized rat models. The in vitro study (cultured osteoclasts) showed that Guizhou Epimedium (E. leptorrhizum Stearn) inhibited the osteoclastic resorption of bone. The in vivo effect of this Chinese herb was also investigated in rats with osteoporosis induced by ovariectomy and the result demonstrated that both the Epime- dium and estradiol were able to increase mineral content and promote bone formation (33). Icariin-containing compounds have been isolated from aerial parts of plants of the genus Epimedium. The icariin-containing compositions were found to be effective in treating osteoporosis. Bone phosphorus and calcium contents, bone mineral density, and the femur strength of treated ovariectomized rats were increased significantly compared to control groups (34). Therefore, herba Epimedii may be of potential use in the treatment of

234 T ABLE 1 Summary of Herba Epimedii and the Immunomodulating Activity

In vitro

Source

Conclusions Li et al.,

Assay

Test substance

Response

Baohuoside I may have 1991 (26)

Various concentrations

Baohuoside I

Baohuoside I has

of baohuoside I were

potential as an incubated with human

significant suppressive

anti-inflammatory/ neutrophils, lymphocytes,

effects on neutrophil

immunosuppressive K562 cells, or gibbon

chemotaxis, mitogen-

agent. leukemic MLA-144 cells

induced lymphocyte

transformation, mixed- lymphocyte culture, NK-cell cytotoxicity and IL-2 production.

Shan et al., Human lymphocytes were

E. brevicornum has 1999 (27)

E. brevicornum

E. brevicornum

immunomodulating extract. Ig & IL production

cultured with epimedium

crude extract

significantly enhance

activity on human detected by ELISA

IgG production by B cells

and stimulate monocytes

lymphocytes in vitro.

to produce IL-1. Animal studies Total

sample

Source Test substance

Conclusions Yap

Iinuma et al., E. sagittatum

ES promotes phagocytic 1990 (25)

Mice

ES, icariin, and

(ES)

epimedin C isolated

activity of and

from ES activated

reticuloendothelial

the phagocytosis of

system in mice, has Yong

carbon by Kupffer

stimulatory effect on

cells in the liver.

macrophage.

Epimedium Li et al.,

The immunosuppressive 1994 (28)

Baohuoside I

Mice

Baohuoside I

suppressed antibody

properties of

and delayed-type

baohuoside I are

hypersensitivity

confined to the

responses in mice in

antibody-mediated

system, suggesting Species

a dose-dependent

fashion. In contrast,

that it might be of

it did not significantly

value in chronic

prolong survival of

inflammatory and

cardiac grafts or

autoimmune diseases

potentiate the effects

treatment.

of the standard antirejection drug cyclosporine.

Liang et al., Flavonol

n-Butanol fraction can 1997 (2)

BALB/c

21 n-Butanol fraction

significantly potentiate from E.

significantly improved

the immune function. hunanense,

C 57 BL/6j

45 the SAFC function in

Epimedin C is an active sc injection

mice

(n = 9)

normal and HCA-

C 57 BL/6j

(n = 6)

suppressed BALB/c

compound of immuno-

mice

mice, significantly

enhancing effects.

recovered lymphocyte proliferation and IL-2 production of HCA-

suppressed C 57 BL/6j mice.

Chen et al., Baohuoside I

Baohuoside I and its 2000 (29)

BALB/c to

Baohuoside I significantly

C 57 BL/10

prolonged mouse heart

analogs useful as

combination

allograft survival. No

antirejection agents in

toxic effects were

organ transplantation.

observed.

T ABLE 1 Continued

1C. Human studies Total

sample

Source Test substance

Conclusions Chen et al.,

ES was effective 1995 (30)

E. sagittatum (ES)

Hemodialysis

In hemodialytic patients,

patients

levels of both sIL-2R

in regulating

and IL-6 could be

immunodeficiency

restored to normal

states in endstage

renal failure patient. Liao et al.,

after treatment with ES.

ES had therapeutic effect 1995 (31)

E. sagittatum (ES)

Hemodialysis

34 IL-2 activity of peripheral

decoction

patients

blood monocytes (PBMC)

on sexual disorder and

stimulated by PHA

immunological

increased significantly in

inadequacy in chronic

patients treated with ES.

renal failure patients on hemodialysis.

Cai et al., E. brevicornum (EB)

EB could relieve 1998 (32)

Patients on

65 Significant differences on

and prednisone

prednisone

ACTH, corticosterone,

neuroendocrino-

and lymphocyte

immunological

proliferative reaction

effect inhibited by

were found between

exogenous

treatment and control

glucocorticoid. Yap

group.

and SAFC: spleen antibody-forming cells; HCA: hydrocortisone acetate.

— indicates unknown. Yong

Epimedium Species 237

osteoporosis. However, the mechanisms of action for prevention of osteopo- rosis were not described. Table 2 summarizes the protective effects of Epi- medium on bone health.

C. Endocrine Effects It has been reported that E. brevicornum may reduce the cold-resistant po-

tential of normal mice and disturb the balance of thyroid hormones in normal rats. Large doses of E. brevicornum may exert an unfavorable effect on normal rats, such as reducing natural weight gain and serum triiodothyronine (T3), but increasing rT3 and thyrotropin-releasing hormone levels as well as raising thyroid-stimulating hormone (40). Lower T3 and higher rT3 levels may be due to peripheral metabolism of thyroid hormones. It was concluded that large doses of this yang-restoring medicine should not be given to the orga- nisms without symptoms of yang deficiency. Another study showed that E. brevicornum could raise plasma corticosterone level significantly and de- creased plasma T3 level in rats, but no rise of plasma testosterone level was observed (41). In a study of 65 patients, the plasma level of adrenocortico- trophin and corticosterone decreased and lymphocyte proliferative reaction was reduced ( p < 0.05) after treatment with E. brevicornum when compared with the control group (32). Epimedium herb was found to inhibit mono- amine oxidase (MAO) in the hypothalamus, leading to higher levels of nor- adrenalin and dopamine (42). The researchers also showed that noradrenalin, adrenaline, serotonin, and dopamine levels were all elevated in animals given Epimedium. Higher levels of dopamine in particular may help to explain the apparent prosexual effect of horny goat weed. High dopamine levels en- courage the release of luteinizing hormone from the pituitary gland, which in turn stimulates the testes to produce more testosterone. Stress is often accom- panied by the excess release of cortical stress hormones, and Epimedium is thought to reduce the effects of corticoids on the body. Generally the herb is believed to have aphrodisiac properties.

D. Antioxidant Effects Methanol extracts of 180 Oriental herbs were screened for their antioxidant

activities by determining the peroxide values of linoleic acid during storage at 50jC (43). Among the herbal extracts tested, 44 selected herbal extracts were further studied in a methyl linoleate system for an extended storage time. Eleven herbs had particularly high antioxidant activities, where the antiox- idative effects of most herbal extracts were greatly dependent on the extrac- tion solvent used. However, E. koreanum Nakai extract and three other herbs appeared to show significantly strong antioxidant activities regardless of the solvents used for the extraction ( p < 0.05). This result suggested that E.

T ABLE 2 Summary of Herba Epimedii and Effects on Bone

In vitro

Source Assay

Conclusions Liu et al.,

Test substance

Response

E. sagittatum polysaccharides 1991

Bone marrow cell cultures

E. sagittatum

Treatment with 100 Ag of

increased cell multiplication (35)

of ‘‘yang deficiency’’

polysaccharides

E. sagittatum polysaccharides

and DNA synthesis of bone by hydroxyurea

animal model caused

increased cell multiplication and

DNA synthesis rate by 72% and

marrow cell cultures

68%, respectively. Animal studies Total

sample

Source Test substance

Conclusions Wu

Subjects

size

Type of study

Response

It can antagonize et al.,

Water extract

Rats

Herba Epimedii

adrenocortical 1996

of herba

can prevent

atrophy and (36)

Epimedii

the side effects

induced by

osteoporosis.

long-term use of glucocorticoids in rats.

Peng E. leptorrhizum

cAMP was not Yap et al.,

Male

E. leptorrhizum has

involved to 1997

Sprague-

no significant

regulate the and (37)

Dawley rats

effects on

endogenous cAMP

mechanism of

in alveolar bone of

E. leptorrhizum Yong

orthodontic tooth.

in promoting bone remodeling.

E. leptorrhizum is Epimedium

Yu E. leptorrhizum

Ovariectomized

Both Epimedium and

et al., Stearn;

of potential use 1999

rats

estradiol were able

in the treatment (33)

estradiol

to increase mineral

content and promote

of osteoporosis.

Epimedium has Species et al.,

bone formation.

Ma Epimedium

Wistar

54 Randomized

Trabecular volume in

some therapeutic 1999

ovariectomized

(n = 10)

total bone volume

effects on (38)

rats

(TV/TBV), and osteoid

percentage increased

osteoporotic rat

significantly after

models.

Epimedium treatment.

Wang Epimedium

Epimedium can et al.,

inhibit the 2000

(OVX) rats

(n = 10)

significantly higher,

expression of (39)

but the IL-6 mRNA

expression level was

IL-6 mRNA, which

significantly lower in

may contribute to

epimedium group than

its antiresorptive

effect. Chen

that in OVX group.

Icariin-containing et al.,

Icariin—

Ovariectomized

Bone phosphorus,

compositions with 2000

standardized

Wistar rats

calcium contents,

20–40% icariin (34)

composition

bone mineral

density, and femur

content by weight

strength of icariin-

are still effective

treated ovariectomized

in treating

rats were increased

osteoporosis.

significantly as compared to control.

— indicates unknown.

240 Yap and Yong

koreanum extract contains powerful antioxidative components. Phenolic acids and/or flavonoids might be the possible antioxidative components because methanol is a good solvent for extracting these compounds from the plant materials. Also, Epimedium compound granules, which lower blood lipid, have anti-free radicals and adjust the balance between prostacyctin I2 and thromboxane A2 in patients with kidney deficiency syndrome of ischemic cardiocerebrovascular diseases (44).

E. Anticancer Effects Epimedium is officially listed in China as an herb that can help prevent the

growth of cancer and has been listed by the Chinese Academy of Medical Sciences as one of an elite group of herbs that slow aging and promote longevity. The plant bioactive compound baohuoside I was shown to have cytotoxic and cytostatic effects on six different cancer cell lines, by means of

microscopy, 51 Cr-release, and growth inhibition. The IC 50 on the cells tested ranged from 2.8 to 7.5 Ag/mL. At tumoricidal concentrations, inhibition of DNA and RNA synthesis, but not protein synthesis, was observed (45). Investigators at the University of Heidelberg published provocative results on herba Epimedii glycoside icariin extract. In vitro experiments showed that this herb stimulated healthy cells to produce an anticancer substance called tumor necrosis factor-a. Also, icariin purified from E. koreanum induced differen- tiation of human promyelocytic leukemic cells (HL-60) by reducing nitroblue tetrazolium and elevating the cAMP/cGMP ratio. There were many rugos- ities and ball-like processes on the cell surface and the mechanism might be related to elevated cAMP/cGMP ratio (46). These preliminary studies suggest that herba Epimedii may be useful against cancer.

F. Antihepatotoxic Effects Icariin compounds display antihepatotoxic activity in cultured rat hepato-

cytes. Icariin was reported to significantly reduce the levels of glutamic pyruvic transaminase and sorbitol dehydrogenase release, decrease glutathi- one-S-transferase (GST) activity, and increase cytochrome P-450 content in

CCl 4 -intoxicated rat hepatocytes (47).

G. Antibacterial and Antiviral Effects Epimedium has been shown to actively inhibit the growth of staphylococci,

streptococci, and pneumococci in culture. Recent studies have shown that Epimedium has significant anti-HIV activity. Epimedium polysaccharides were found to reverse the zidovudine (AZT)-induced inhibition of DNA synthesis and reduce the toxicity of AZT (48).

Epimedium Species 241

H. Cardiovascular Effects Some of the flavonol glycosides and flavonoids isolated from the genus are

pharmacologically effective in dilation of the coronary artery, inhibition of platelet aggregation, and delayed formation of thrombi (8). Chen et al. (49) described the isolation of five new prenyflavones, yinyanghuo A, B, C, D, and

E. Yingyanghuo A and B were shown to have significant antiplatelet activity. Flavone glycosides of E. koreanum (TFG) stimulated murine macrophages. The fibrinolytic activity of stimulated macrophages was approximately 2.8-fold that of the controls. In vivo experiments reported that the effect of TFG on spontaneously hypertensive and apoplexic rats was very evident. The abiotic rate of TFG-stimulated rats was 5%, while that of the control group was 90% (50). Epimedium may help to dilate the coronary vessels, increasing coronary blood flow by reducing vascular resistance (51). E. decoctum and its extracts give certain protection in the myocardial ischemia of rat caused by pituitrin. In rabbits, intravenously injected icariin (1 mg/kg) and water extract of E. grandiflorum containing icariin at a dose equivalent to 1 g plant/kg did not significantly change the heart rate and electrocardiogram. Other myo- cardial and circulatory indices indicated that both icariin and the extract decreased peripheral resistance, suggesting their potential use in the treatment of hypertension-complicated coronary diseases.

I. Effects on the Reproductive System Epimedium has been reported to have proreproductive effects in the popular

press. Teeguarden (52) anecdotally reported that E. sagittatum seemed to stimulate the sensory nerves throughout the body, particularly in the genital region. The effect of Epimedium herb on the testes, prostate gland, and leva- tor ani muscle (which supports the body in thrusting movements) was re- ported to enhance male sexual function indirectly (53). E. sagittatum appeared to have a sexual potentiation effect and to improve the quality of life in the patients of chronic renal failure with regular hemodialysis (39). Icariin from Epimedium could inhibit contraction of arterial smooth muscle rings that restrict blood flow in the body, via a Ca 2+ channel-blocking mechanism, further enhancing vasodilation (48). On the other hand, the chief ingredient of Epimedium herb, icariin, can promote the development of epididymes and seminal vesicle of mouse. In vitro animal studies suggest that icariin may enhance the production of testosterone (42).

J. Urinary Effects Epimedium may improve immunological deficiencies in chronic renal fail-

ure patients on dialysis, and was reported to significantly improve blood

242 Yap and Yong

urea nitrogen and serum creatinine levels in rats. In the gromeruli, it was reported to inhibit deposition of IgG and C3 along glomerular capillary walls in rats (54). Tan and Weng (44) reported on the efficacy of Epimedium com- pound pills in the treatment of ‘‘kidney-deficiency syndrome’’ of ischemic cardiocerebro vascular diseases. The rates of improvement were reported to

be 70% in the electrocardiogram of the patients with coronary heart dis- eases, and 75% in the electroencephalogram of the patients with cerebral atherosclerosis.

K. Respiratory Effects The herb acts as an antitussive and expectorant. Studies of its effectiveness in

1066 cases of chronic bronchitis demonstrated a 74.6% effectiveness rate in improving symptoms (48). It was effective in protecting guinea pigs from histamine-induced asthma.

L. Others Methanol extracts of E. sagittatum were found to induce significant neurite

outgrowth activity on cultured PC12H cells. Bioassay-guided fractionation yielded six prenylated flavonol glycosides, ikarisoside A, icarisid II, epimedo- side A, icariin, epimedin B, and epimedokoreanoside-1, as the active ingre- dients (3). A recent study also demonstrated that two syringaresinol enantiomers isolated from E. koreanum Nakai and Magnolia officinalis Rehd, as well as a mixture of their glucosides, showed dose-dependent neurito- genesis in a concentration range from 0.24 to 24 AM in PC12h cells (55). E. grandiflorum has also been used to treat hot flashes in complex mixtures of

8 (56) or over 20 herbs (57).

V. ADVERSE EFFECTS AND TOXICITY Epimedium is a safe and benign herb. There is no record of adverse side

effects, toxicity, or contraindications to taking Epimedium by humans. In some people, ingesting it can lead to dizziness, thirst, dry mouth, vomiting, and nosebleed.

VI. SUMMARY The use of traditional Chinese concepts like yin/yang and qi, which have no

modern physiological equivalents, in chinese publications make scientific interpretation of these studies difficult. Problems are compounded by the use of terms such as ‘‘kidney’’ and ‘‘spleen,’’ which often do not have the same meaning as understood in the Western medical literature. Herbs are used to

Epimedium Species 243

treat yin/yang and qi deficiency states with no clear outcome parameters. Nevertheless these studies indicate that Epimedium herb may have biological activities that can affect cell function and therefore health status. However, the effective doses, pharmacokinetics, and cellular mechanisms have not yet been proven by science or substantiated through long-term clinical research. Owing to the limited data, no firm conclusions can be made at this time. It is critical to determine exactly which mechanisms operate in which circum- stances and how these components work in view of the widespread use of this herb.

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