Chinese Herbs increase ATP production and antioxidants

Aging
is a process of bodily change with time, leading to increased
susceptibility to disease, and ultimately death. Because reactive
oxidant species (ROS) and immune dysfunction are major causes of
age-related diseases [1-3],
the maintenance of antioxidant and immune fitness is a rational
approach to preventive health care. Accumulation of ROS-induced
oxidative damage to DNA, proteins, and other macromolecules has been
regarded as a major endogenous cause of aging [1].
In addition to ROS-mediated cellular damage, aging was found to be
associated with immune senescence, attributable at least partly to the
loss of T lymphocyte functions [2,3].
Such loss increases the prevalence of infectious diseases in the
elderly. With advances in modern medical research techniques, research
on age-related chronic illnesses has become intense, in the quest for
valuable preventive and therapeutic measures. Humans have been making
continuous efforts to fight aging. As Chinese medicine has always
emphasized the prolongation of a healthy lifespan, many Chinese
tonifying herbs have long been used to safeguard health and to delay
the onset of senility.

Under both normal and pathological
conditions, ROS are generated in all cells undergoing aerobic
metabolism, particularly from mitochondria. The cell possesses two
distinct antioxidant defense systems to counteract damaging ROS: (1)
enzymatic antioxidants such as catalase, superoxide dismutase (SOD),
glutathione peroxidase and other related enzymes/molecules, and (2)
non-enzymatic antioxidants such as ascorbic acid (vitamin C),
α-tocopherol (vitamin E) and β-carotene. To achieve optimal antioxidant
fitness, every component of the antioxidant defense system should
function optimally because antioxidants must work together in a
synergistic manner. Chinese tonifying herbs have been shown to possess
both in vitro and in vivo antioxidant activities [4,5].

The
immune system fights against 'foreign invaders' such as bacteria,
viruses, fungi, yeasts and parasites. The humoral and cell-mediated
immune responses show great competence in dealing with intruders.
Moreover, the surveillance function of the immune system tends to
prevent cancers, particularly in old age. However, an overreactive or
imbalanced immune system can cause allergies or autoimmune disorders. A
well-constituted and balanced immune system is thus crucial for
safeguarding health. Chinese tonifying herbs have been shown to
stimulate or suppress the cell-mediated immune response both in vitro and in vivo [6].

The
importance of disease prevention has been recognized by Chinese
medicine through experience accumulated over centuries. Many Chinese
tonifying herbs have long been used for safeguarding health and for
delaying the onset of senility. According to Chinese medicine theories,
tonifying herbs prescribed for various symptoms of ill-health are
generally classified into four categories on the basis of their
health-promoting actions, namely 'Yang-invigorating', the 'Qi-invigorating', the 'Yin-nourishing' and the 'Blood-enriching' herbs [7]. The 'Qi-invigorating'
and 'Blood-enriching' herbs are of Yang and Yin characteristics
respectively. Chinese medicine theories suggest that a balance of Yin
and Yang is essential to sustain optimal body function [8].
From a modern medical perspective, the maintenance of Yin and Yang in
harmony may be described as the attainment of bodily homeostasis. The
long-known antagonistic relationship between parasympathetic and
sympathetic neural activities affords an example of both a phenomenon
well-recognized by Western medicine and the Yin/Yang balance. A recent
psychophysiological investigation in humans revealed an association
between decreased parasympathetic or sympathetic activities with
deficiencies of Yin or Yang respectively [9].

The
theoretical framework of Chinese medicine is based on the Chinese
cultural fabrics and clinical experience, while modern Western medicine
has been established on the basis of laboratory and clinical
investigations [10].
As the two distinct medical systems are complementary, bridging of the
knowledge gap between Chinese and Western medicine is essential for
their integration, in clinical practice, for disease prevention and
treatment. Expounding Chinese medicinal theories in modern scientific
terms to a Western audience facilitates communication between
practitioners of the two systems.

In our earlier studies, we
found that tonifying herbs with Yang or Yin properties were associated
with antioxidant and immunostimulatory activities respectively [4].
Recent studies indicated that only Yang tonifying herbs (not Yin
tonifying herbs) enhanced mitochondrial ATP generation capacity in
mouse hearts [11].
We therefore suggest that Yang tonifying herbs enhance mitochondrial
ATP generation, while Yin tonifying herbs are associated with
immunomodulatory activities. In this mini-review, we summarize the
abilities of Yang and Yin tonifying herbs to enhance ATP generation
capacity, and to potentiate antioxidant and/or immunomodulatory
actions, in an effort to characterize their respective pharmacological
properties.

In
Chinese medicinal theories, Yang is a manifestation of body functions
supported by various organs. A 'Yang-invigorating' action therefore
involves the enhancement of bodily functions in general and cellular
activities that consume ATP in particular. The mitochondrion is
responsible for the generation of ATP through oxidative metabolism. To
establish the pharmacological basis of 'Yang-invigorating' action, we
have recently investigated the effect of Yang herbs on ATP generation
capacity in heart homogenates prepared from mice that were pretreated
with methanolic extracts of herbs [11].
Tonifying herbs from other functional categories were examined for
comparison. While Chinese herbs are usually extracted by water for
human oral consumption, water was replaced by methanol in our study for
convenience in the processing and storage of samples. Yang herbs
invariably enhanced myocardial ATP generation, with stimulation ranging
from 20–130%. Herba Cynomorii and Semen Cuscutae were
the most potent herbs examined. By contrast, none of the Yin herbs
enhanced ATP generation; some Yin herbs even suppressed ATP generation
slightly (Table 1).
A preliminary mechanistic study indicated that Yang herbs may speed up
ATP synthesis by increasing mitochondrial electron transport [11].

Mitochondrial
oxidative phosphorylation generates ROS as byproducts. Highly reactive
chemically, ROS attack cellular structures located near the sites where
ROS are generated. Mitochondrial DNA, proteins, and lipids in the inner
membrane of mitochondria are thus vulnerable to oxidative damage [12], resulting in generalized organelle dysfunction, defective mitochondrial biosynthesis and poor energy metabolism [13].

Under
normal physiological conditions, the mitochondrial antioxidant defense
system adequately handles the potentially detrimental effects of ROS
derived from energy metabolism [14].
When a functional imbalance between ROS levels and antioxidant
concentrations caused by various disease states and/or aging occurs,
age-related disorders such as cancer, cardiovascular diseases, brain
dysfunction, or cataract may occur [15]. Antioxidant supplementation, particularly from herbal extracts, has become a trend in preventive health care.

Using an oxygen radical absorbance capacity assay, Ou et al. recently compared the free radical scavenging (i.e. antioxidant) activities of Yang and Yin herbs [16].
The results indicated that Yin herbs generally possessed higher
antioxidant activities than Yang herbs and that the antioxidant
potencies correlated well with the amounts of total phenolic compounds
in the herbs. The authors suggested an analogy between Yin/Yang balance
and antioxidation/oxidation in energy metabolism. These findings of
higher antioxidant activities in Yin herbs as compared with those in
Yang herbs do not agree with the findings from one of our earlier
studies which showed that most of the Yang herbs possessed a more
potent 1,1-diphenylpicryhydrazyl radical-scavenging action than other
tonifying herbs [4] (Table 2).
Although the use of different herbal extraction methods and distinct
antioxidant assays precludes direct comparison of the two studies, the
discrepancy might be due to the selection of almost completely
different sets of Yin and Yang herbs for testing in the two studies.
Our study focused on herbs used for safeguarding health (i.e. herbs
used for tonifying purposes) (Tables 2, Table 3 of reference [17]). Ou et al.
probably used a selection criterion based on the general Yin and Yang
properties of the herbs instead of their Yin-tonifying and
Yang-tonifying actions [16]. Szeto and Benzie, using the same set of herbs described in Ou et al.
to examine possible protective effects on DNA oxidative damage, found
that the Yang herbs showed an antioxidant effect superior to that of
Yin herbs [5].

Several Yang herbs have been shown to possess antioxidant activities both in vitro and in vivo (Table 4). In vitro free radical-scavenging activities were detected in herbal extracts prepared from Herba Epimedii [4,18], Radix Dipsaci [4,16], Fructus Psoraleae [4], Semen Cuscutae [16], Herba Cistanche [4,16,18], Cortex Eucommiae [19] and Rhizoma Cibotii [4,16]. Aqueous extracts of Rhizoma Drynariae and Cortex Eucommiae were found to inhibit oxidant production from rat osteoblasts [20], and also inhibited biomolecular oxidative damage [21]. Active ingredients (bakuchiol, isobavachin and isobavachalcone) from Fructus Psoraleae inhibited the NADPH-dependent peroxidation of rat microsomal and mitochondrial lipids in vitro [22]. An ethanolic extract of Radix Dipsaci enhanced the antioxidant status of blood and liver in rodents [23] and a Radix Morindae extract increased blood antioxidant enzyme activities in diabetic rats [24]. Phenylethanoids isolated from Herba Cistanche were found to prevent cell damage induced by in vitro and in vivo exposure to carbon tetrachloride in rats [25]. A recent study from our laboratory indicated that pretreatment with the methanolic extract of Herba Cistanche protected against ischemia-reperfusion injury in rat hearts ex vivo and enhanced mitochondrial ATP generation in the rat hearts ex vivo and H9c2 cells in situ.
The ATP-stimulating action was possibly due to enhanced oxidative
phosphorylation caused by increases in the activities of complexes I
and III [26].
As good body function requires a large amount of energy and antioxidant
defense is essential in sustaining mitochondrial ATP production [27],
the antioxidant activities of Yang herbs may safeguard ATP generation,
particularly under conditions of upregulated cellular activities.

Methanolic extracts of both Fructus Ligustri and Herba Ecliptae were found to enhance hepatic glutathione (GSH) regeneration capacity in rats [4,28]. The enhancement of hepatic GSH regeneration capacity by Fructus Ligustri was associated with a hepatoprotective action against carbon tetrachloride toxicity [28]. Activity-directed fractionation of Fructus Ligustri indicated
that the hepatoprotective principle(s) resided mainly in the oleanolic
acid-enriched butanol and chloroform fractions [28].
Moreover, our recent studies showed that both short and long term
pretreatment with oleanolic acid protected against myocardial
ischemia-reperfusion injury in rats [29,30].
It was suggested that the cardioprotection afforded by oleanolic acid
pretreatment was related to the enhancement of mitochondrial
antioxidant mechanism mediated by GSH and α-tocopherol [29].
Both experimental and clinical investigations indicated that the
antioxidant status influenced immunocompetence, particularly under
conditions of stress such as physical exercises or chronic diseases [31]. The antioxidant activities of Yin tonifying herbs may positively influence immunostimulatory activities.

A 'Yang-invigorating' herbal formula named VI-28 has been shown to produce 'Yang-invigorating' effects [32]
and enhance red cell antioxidant status, particularly Cu-Zn-superoxide
dismutase (SOD) activity, in elderly male human subjects [33]. This herbal formula is comprised of Radix Ginseng, Cornu Cervi, Cordyceps, Semen Allii, Fructus Cnidii, Fructus Evodiae and Rhizoma Laemferiae.
Recently we investigated the effects of long-term VI-28 treatment on
red cell Cu-Zn-SOD activity, mitochondrial functional ability, and
antioxidant levels, in various tissues of rats of both sexes [34].
The results indicated that VI-28 treatment increased red cell Cu-Zn-SOD
activity and mitochondrial ATP generation capacity, increased the
levels of reduced GSH and α-tocopherol, and reduced Mn-SOD activities.
The enhancement of ATP generation by VI-28 increased mitochondrial ROS
production, resulting in the upregulation of mitochondrial antioxidant
mechanism. The VI-28-induced increase in mitochondrial antioxidant
capacity in various tissues was evidenced by a significant reduction in
ROS generation. Given that cellular energy status and mitochondrial ROS
generation are factors critically involved in aging, the dual effect of
'Yang-invigoration' produced by VI-28 may have clinical implications in
the prevention of age-related diseases.

It
was suggested that the proper functioning of the immune system requires
dynamic interactions between Yang and Yin. And while the
antigen-nonspecific immune response is associated with Yang, the
antigen-specific response is related toYin [35].
One of our earlier studies investigated antioxidant and
immunomodulatory activities in different categories of tonifying herbs.
The results showed that 6 and 7 of a total of 8 Yin herbs tested
potentiated concanavalin A (Con A)-stimulated splenocyte proliferation
(an antigen-specific response) in mice in vitro and ex vivo respectively. By contrast, only 3 of 9 Yang herbs tested showed a similar enhancement of the Con A-stimulated immune response [4] (Table 2).

Among the Yin herbs, the methanolic extract of Fructus Ligustri yielded the most robust immunostimulatory action in mouse splenocytes [4]. Differential extraction of Fructus Ligustri by
solvents of increasing polarity indicated that the immunostimulatory
activity resided mainly in the petroleum ether fraction [36]. Oleanolic acid, an immunomodulatory triterpenoid commonly found in herbs including Fructus ligustri [37,38], was undetectable in this fraction [36]. Currently, activity-directed fractionation of the petroleum ether extract of Fructus Ligustri is
under way in our laboratory. Various immunomodulatory actions of Yin
tonifying herbs, and the active ingredients of the herbs, have been
reported in other studies (Table 5). An aqueous extract of Radix Asparagi was
found to inhibit tissue necrosis factor-α (TNF-α) secretion by
suppressing Interleukin (IL)-2 secretion from astrocytes, implicating
that the extract might exhibit anti-inflammatory activity in the
central nervous system [39]. Both the crude aqueous extract and the two active ingredients (ruscogenin and ophiopogonin D) of Radix Ophiopogonis produced anti-inflammatory effects in rodents [40].
While the aqueous extract inhibited xylene-induced ear swelling and
carrageenan-induced paw edema in mice, it also suppressed
carrageenan-induced pleural leukocyte migration in rats, and the
zymosan-evoked migration of peritoneal total leukocytes and neutrophils
in mice. Treatments with ruscogenin and ophiopogonin D decreased
zymosan-induced peritoneal leukocyte migration in mice and reduced the
phorbol-12-myristate-13 acetate-induced adhesion of HL60 cells to
ECV304 cells [40]. Several sesquiterpenes isolated from Herba Dendrobii were
found to exhibit immunomodulatory activity by exerting comitogenic
effects on Con A and lipopolysaccharide-stimulated mouse splenocytes [41,42]. It has recently been reported that an ethanolic extract of black rice (the fruit of Oryza sativa) showed anti-asthmatic effects in a mouse model [43].
Treatment with the ethanolic extract of black rice reduced the number
of eosinophils in bronchoalveolar lavage fluid, alleviated the airway
hyper-response, and decreased the extent of airway inflammation in
ovalbumin (OVA)-immunized and -aerolized mice challenged with OVA.
Moreover, the ethanolic extract treatment decreased interferon-γ
(INF-γ), IL-4, IL-5 and IL-13 levels in the supernatants of cultured
splenocytes and suppressed the plasma levels of OVA-specific
immunoglobulin (Ig)G, IgG2α, IgG1 and total IgE in OVA-immunized and
-challenged mice [43]. Clinical investigations indicated that intramuscular injection of undiluted Fructus Ligustri extract at a dose of 2–4 ml once or twice daily could prevent leucopenia caused by chemotherapy or radiotherapy. Fructus Ligustri treatment normalized white blood cell counts, thereby increasing tolerance to chemo/radiotherapy [44]. Oral administration of Fructus Ligustri tablets at a daily dose of 50 g equivalence of crude herb was found to ameliorate the symptoms of chronic bronchitis [44]. A herbal formula comprising Fructus Ligustri, Radix Scutellariae, Radix Astragalus and Eupolyphaga et polyphae was found to alleviate symptoms and improve immune function in HIV/AIDS patients [45].

Ganoderma,
another Yin tonifying herb with immunomodulatory effects, is widely
consumed by the Chinese people who believe that it promotes health and
longevity, lowers the risk of cancer and heart diseases and boosts the
immune system [46]. In Chinese medicine, Ganoderma is regarded as a very potent herb for 'Fu Zheng', a Chinese medicine concept comparable to immunotherapy/immunomodulation in Western medicine. While Ganoderma is
traditionally used to increase the resistance of the body immune system
to pathogens and to restore normal body functions, the herb has now
also been used to decrease the side effects of Western medical
procedures, such as surgery, radiotherapy and chemotherapy which often
weaken the immune system. The anti-cancer/immunomodulatory effects of Ganoderma were associated with triterpenes [47], polysaccharides [48,49] or immunomodulatory proteins [50] through mechanisms involving inhibition of DNA polymerase [51], inhibition of post-translational modification of the Ras oncoprotein [52] or the stimulation of cytokine production [53]. Recent studies on the immunomodulatory activities of Ganoderma indicated that Ganoderma extract
stimulated the proliferation of human peripheral blood mononuclear
cells and raised the levels of mRNAs encoding Th1 and Th2 cytokines in
these cells [54]. Moreover, polysaccharides of Ganoderma activated
mouse splenic B cells and induced these cells to differentiate into
IgM-secreting plasma cells. This process was dependent on the
polysaccharide-mediated induction of Blimp-1, a master regulator
capable of triggering a cascade of gene expression during plasmacytic
differentiation [55]. In human peripheral B lymphocytes, the Ganoderma polysaccharide
fraction enhanced antibody secretion and induced the production of
Blimp-1 mRNA, though it failed to induce lymphocyte differentiation [55].

In addition to immunomodulating activities, Ganoderma possesses in vivo antioxidant potential, another aspect of Yin tonifying action. Treatment with Ganoderma extract was found to enhance the hydroxyl radical scavenging activity of rabbit blood plasma [56,57]. Ganoderma acted by stimulating cellular and mitochondrial SOD activities, thereby enhancing the antioxidant capacity of the body [58]. It was shown that an intraperitoneal injection of Ganoderma extract following a lethal dose of cobalt X-ray radiation caused a marked prolongation of survival time in mice [59]. Pretreatment with Ganoderma extract
also markedly protected against carbon tetrachloride-induced hepatic
damage and the associated impairment in hepatic antioxidant status [60].

Cordyceps,
a premium Chinese tonifying herb which replenishes the 'kidney' and
soothes the 'lung', is prescribed for the treatment of a host of
disorders, including hyposexualities, hyperglycemia, hyperlipidemia,
asthenia after illness, respiratory diseases, renal disorders, liver
and heart diseases [61]. Cordyceps is
regarded as a tonifying herb with both 'Yin-nourishing' and
'Yang-invigorating' actions. Pharmacological studies have shown that Cordyceps possesses a wide spectrum of biological activities including antioxidation [61-64], immunopotentiation [65-68], anti-tumorigenesis [68-71], anti-inflammation [72] and stimulation of testosterone biosynthesis [73]. We have recently investigated the effects of wild and cultured Cordyceps on Con A-stimulated splenocytes (an in vitro bioassay for Yin tonifying action) and myocardial ATP generation capacity (an ex vivo bioassay for Yang tonifying action) [74]. The results indicated that methanolic extracts of wild and cultured Cordyceps enhanced both the Con A-stimulated splenocyte proliferation in vitro and myocardial mitochondrial ATP generation ex vivo in mice, with no significant difference in potencies when the two types of Cordyceps were
compared. While the immunopotentiating effect was associated with an
increase in IL2 production, the stimulation of myocardial ATP
generation was paralleled by an enhancement in mitochondrial electron
transport. When compared with typical Yin and Yang tonifying herbs (Fructus Ligustri and Herba Cynomorii respectively), Cordyceps was
found to possess both Yin and Yang tonifying actions, with a lower
potency in both modes of action. The observation of both
immunopotentiating and ATP-enhancing activities in Cordyceps extracts further supports the pharmacological basis of Yin and Yang tonifying herbs in Chinese medicine.

Yang
tonifying herbs stimulate mitochondrial ATP generation, leading to the
enhancement of cellular/mitochondrial antioxidant status, presumably
through the intermediacy of ROS. Yin tonifying herbs, which also
possess antioxidant properties, are mainly immunomodulatory, thereby
boosting weak immune functions and suppressing overreactive or
unbalanced immune responses. Cordyceps, highly regarded as a
tonifying herb with a dual action of Yin and Yang, stimulates
mitochondrial ATP generation and enhances cellular immune responses.
Given that impairment in mitochondrial functional ability and
antioxidant status, and a decline in immunocompetence, are believed to
be critically involved in the development of age-related diseases and
the aging process, the abilities of Yang and Yin tonifying herbs to
enhance ATP generation capacity and to produce antioxidant and
immunomodulatory actions are beneficial for safeguarding health and
delaying the onset of senility (Figure 1).
While animal models may be used for testing working hypotheses on Yang
and Yin tonifying actions, clinical studies, using Yang and Yin
tonifying herbs and/or defined chemicals isolated from the herbs or
synthesized in the laboratory, on age-related variations in antioxidant
and immune function, would be of considerable value.