K̲ai-X̲in-S̲an (KXS), originated from traditional Chinese medicine (TCM), is a herbal formula that has been used for treating mental disorders, especially memory loss and depression, in China for thousands of years. KXS has a combination of four herbs: Ginseng Radix et Rhizoma (GR; root and rhizome of Panax ginseng C. A. Mey.; Araliaceae family), Polygalae Radix (PR; root of Polygala tenuifolia Wild.; Polygalaceae family), Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott; Acoraceae family), and Poria (PO; sclerotium of Poria cocos (Schw.) Wolf; Polyporaceae family). KXS contains two paired-herbs: GR-PR aiming at activating ”Xin-qi”, i.e., the benefit of brain functions and enhancing memory; ATR-PO aiming at eliminating dampness and inducing resuscitation, i....[ Read more ]

K̲ai-X̲in-S̲an (KXS), originated from traditional Chinese medicine (TCM), is a herbal formula that has been used for treating mental disorders, especially memory loss and depression, in China for thousands of years. KXS has a combination of four herbs: Ginseng Radix et Rhizoma (GR; root and rhizome of Panax ginseng C. A. Mey.; Araliaceae family), Polygalae Radix (PR; root of Polygala tenuifolia Wild.; Polygalaceae family), Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott; Acoraceae family), and Poria (PO; sclerotium of Poria cocos (Schw.) Wolf; Polyporaceae family). KXS contains two paired-herbs: GR-PR aiming at activating ”Xin-qi”, i.e., the benefit of brain functions and enhancing memory; ATR-PO aiming at eliminating dampness and inducing resuscitation, i.e. functions related to brain function. These two paired-herbs are contributing to KXS clinical efficiency, and different compatibilities of paired-herbs are leading to three KXS, i.e. KXS-652 formula of GR:PR:ATR:PO as 1:1:25:50; KXS-984 of GR:PR:ATR:PO as 1:1:1:2; DZW-652 of GR:PR:ATR:PO as 3:2:2:3 (the number indicated the year it was created).

In previous study, different compatibilities of GR-PR and ATR-PO showed diverse actions on neuronal differentiation that is considered as a crucial target for anti-depression. Thus, there might be an optimal ratio of paired-herbs to form a new patentable herbal combination showing promising effects on anti-depression. Here, we aimed to prepare an optimized formula by searching for the compatibility of paired-herbs on neuronal differentiation. Whether the new combination possessed promising effects on anti-depression by other therapeutic targets, e.g. expression of neurotrophic factors and synapse formation would be further tested.

To explore an optimized ratio of paired-herbs in KXS, the chemical and biological assessments of single herbal extract, paired-herb extracts and KXS extract were determined and compared. Cultured pheochromocytoma (PC12) cells were used to study the effects of herbal extracts on neuronal differentiation, where neurofilaments were used as indicative markers. By revealing the herbal extract-induced transcriptional activity of neurofilaments, an optimized ratio (named KXS₂₀₁₂, the number indicated the year it was created) of paired-herbs was revealed: the significant synergistic effects of KXS₂₀₁₂ were analyzed by Compusyn, a computer program in examining drug interaction in multiple drug effect analysis according to median-effect principle.

Several cell models were employed to study the effects of KXS₂₀₁₂ on three anti-depressive targets: neuronal differentiation, synaptogenesis and neurotrophic factor expression. In neuronal differentiation, KXS₂₀₁₂ showed better effect, as compared to KXS, in promoting differentiation of cultured PC12 cells, including the potentiation of nerve growth factor (NGF)-induced neurite outgrowth and neurofilament expression. The potentiating effect of KXS₂₀₁₂ might be mediated through NGF receptor, tropomyosin receptor kinase A (Trk A): because the receptor expression was markedly increased in KXS₂₀₁₂-treated PC12 cells, and the potentiating effect was inhibited by K252a (an inhibitor of tyrosine phosphorylation of Trk A). In synaptogenesis study, KXS₂₀₁₂ showed better effect on inducing expression of synaptotagmin than that of KXS. In addition to induce the expression levels of NGF, brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), KXS₂₀₁₂ greatly induced the expression of neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5), as compared to KXS. The induction of neurotrophic factors was inhibited by H89 [Protein kinase A (PKA) inhibitor] and U0126 [Mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK1/2) inhibitor], indicating that KXS or KXS₂₀₁₂-induced neurotrophic factor expression was mediated through cAMP-PKA and MAPK signaling pathways.

At last, depressive animal models were employed to reveal the efficacy and action mechanism of KXS₂₀₁₂. In ICR mice, KXS₂₀₁₂ reduced cumulative immobility time in forced swimming and tail suspension tests. In CMS-induced depressive SD rat model, KXS₂₀₁₂ reversed depressive behavior and up-regulated the levels of neurotransmitters and neurotrophic factors in depressive brain. Compared with KXS, KXS₂₀₁₂ largely restored depressive behavior; increased the level of dopamine in striatum; up-regulated the expression of NGF, NT4/5, Trk B and Trk C in CMS-induced depressive rats. The results indicated that KXS₂₀₁₂ might exert robust anti-depressive function by further up-regulating neurotransmitters and neurotrophic factors in depressive brain.

In conclusion, KXS₂₀₁₂ exerted anti-depressant function in animals by modulating neurotransmitters, neuronal differentiation, synaptogenesis and neurotrophic factor expression. Thus, KXS₂₀₁₂ could be developed as a new patentable regimen for anti-depression.